AU2016301718B2

AU2016301718B2 - Novel anti-PD-L1 antibodies

Info

Publication number: AU2016301718B2
Application number: AU2016301718A
Authority: AU
Inventors: Zhisheng CHEN; Jing Li; Yong Zheng
Original assignee: Wuxi Biologics Shanghai Co Ltd; Wuxi Biologics Ireland Ltd
Current assignee: Wuxi Biologics Shanghai Co Ltd; Wuxi Biologics Ireland Ltd
Priority date: 2015-08-06
Filing date: 2016-08-05
Publication date: 2022-05-12
Anticipated expiration: 2036-08-05
Also published as: LT3332006T; JP7066614B2; HK1257164A1; FI3332006T3; US12180282B2; SA518390862B1; FR24C1056I2; RS64977B1; IL256803B; FR24C1056I1; SI3332006T1; AU2016301718A1; US20200140554A1; IL256803A; SMT202300487T1; EP3332006A4; BR112018002428A2; EP3332006A1; RU2736151C2; RU2018107658A

Abstract

The present disclosure provides monoclonal antibodies against protein programmed cell death 1 ligand (PD-L1), which can block the binding of PD-L1 to PD-1, and therefore block the inhibitory function of PD-L1 on PD-1 expressing T cells. The antibodies of disclosure provide very potent agents for the treatment of multiple cancers via modulating human immune function.

Description

NOVEL ANTI-PD-LI ANTIBODIES HELD OF THE INVENTION

[0001] The present disclosure generally relates to novel anti-PD-L1 antibodies.

BACKGROUND

100021 Increasing evidences from preclinical and clinical results have shown that targeting immune checkpoints is becoming the most promising approach to treat patients with cancers. Programmed cell death 1, one of immune-checkpoint proteins, play a major role in limiting the activity of T cells that provide a major immune resistance mechanism by which tumor cells escaped immune surveillance. The interaction of PD-1 expressed on activated Tcells, and PD-LI expressed on tumor cells negatively regulate immune response and damp anti tumor immunity. Expression of PD-L1 on tumors is correlated with reduced survival in esophageal, pancreatic and other types of cancers, highlighting this pathway as a new promising target for tumor immunotherapy. Multiple agents targeting PD-i pathway have been developed by pharmaceutical companies, such as Bristol-Myers Squibb (BMS), Merck, Roche and GlaxoSmithKline (GSK). Data from clinical trials demonstrated early evidence of durable clinical activity and an encouraging safety profile in patients with various tumor types. Nivolumab, a PD-l drug developed by BMS, is being put at center stage of the next generation field. Now in 6 late-stage studies, the treatment spurred tumor shrinkage in three of 5 cancer groups studied, including 18% of 72 lung cancer patients, close to a third of 98 melanoma patients and 27% of 33 patients with kidney cancer. Developed by Merck, lambrolizumab is a fully human monoclonal IgG4 antibody that acts against PD-1, which grabbed the FDA's new breakthrough designation after impressive IB data came through for skin cancer. The results from a phase IB study have shown an objective anti-tumor response in 51% of 85 cancerpatients, and a complete response in 9% of patients. Roche's experimental MPDL3280A demonstrated an ability to shrink tumors in 29 of 140(21%) advanced cancer patients with various tumor sizes.

[0003] [However, the existing therapies may not be all satisfactory and therefore new anti PD-1 antibodies are still needed.

BRIEF SUMMARY OF THE INVENTION

[0004] The present disclosure provides novel monoclonal anti-PD-Li antibodies (in particular fully human antibodies), polynucleotides encoding the same, and methods of using the same.

[0005] In one aspect, the present disclosure provides isolated monoclonal antibodies or antigen binding fragments thereof, which are capable of specifically binding to human PD-L at a Kd value no more than 104 M (e.g. no more than <9x 0 4°M., 1 0 8xI M, <7x104° M, <6xI0' M, <5x10"° M, 4xl0°' M, <3xI0"M, <2x104° M, or 10 M) as measured by plasmon resonance binding assay.

[0006] In certain embodiments, the antibodies or antigen binding fragments thereof bind to monkey PD-Li at an EC50 of no more than 10 nM (e.g. no more thanI nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 02nM, 0.nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM, or 0.01nM. In certain embodiments, the antibodies and antigen-binding fragments thereof do not bind to mouse PD-Ll but bind to monkey PD-L1 with a binding affinity similar to that of human PD-Li. In certain embodiments, the antibodies or antigen binding fragments thereof potently inhibit binding of human or monkey PD-L to its receptor (e.g. PD-I), at an IC5( of no more than 100 nM (eg. no more than 50nM, 40nM, 30nM, 20n, 10 nM, 9nM, 8nM, 7nM, 6nM, nM, 4nM, 3nM, 2nM,1 InM, 0.9nM,0.8nM,0.7nM,0.6nM,0.5nM,0.4nM,0.3nM,0.2nM,or 0minM). Incertain embodiments, the EC50 or IC50 is measured by fluorescence-activated cell sorting (FACS) analysis.

100071 In certain embodiments, the antibodies or antigen binding fragments thereof have substantially reduced effector function. In certain embodiments, the antibodies or antigen binding fragments thereof do not mediate ADCC or CDC or both.

[0008] In certain embodiments, the antibodies or antigen binding fragments thereof provided herein comprise a heavy chain CDR sequences selected from the group consisting of: SEQ ID NOs: 1, 3, 5, 13, 15, 17, 25, 27, 29, 37, 39 and 41.

[0009] In one aspect, the antibodies or an antigen binding fragments thereof provided herein comprise a light chain CDR sequences selected from the group consisting of: SEQ D NOs: 7, 9, 11, 19, 21, 23, 31, 33 and 35.

[00101 In certain embodiments, the antibodies or antigen binding fragments thereof provided herein comprise at least one, two, three, four, five or six CDRsselected from the group consisting of: SEQ ID NOs: 1, 3, 5, 7, 9, and 11; or selected from the group consisting of: SEQ ID NOs: 13, 15, 17, 19, 21 and 23; or selected from the group consisting of: SEQ ID NOs: 25, 27, 29,31, 33 and35; or selected from the group consisting of: SEQ ID NOs: 37, 39, 41, 19, 21, and 23.

[0011] In certain embodiments, the antibodies or antigen binding fragments thereof provided herein comprise a heavy chain variable region selected from the group consisting of:

a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5; b) a heavy chain variable region comprising SEQ ID NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 17; c) a heavy chain variable region comprising SEQ ID NO: 25, SEQ ID NO: 27, and/or SEQ I) NO: 29; and d) a heavy chain variable region comprising SEQ ID NO: 37, SEQ ID NO: 39, and/or SEQ ID NO: 41.

[0012] In certain embodiments, the antibodies or antigen binding fragments thereof provided herein comprise a light chain variable region selected from the group consisting of:

a) a light chain variable region comprising SEQ I) NO: 7, SEQ I) NO: 9, and/or SEQ ID NO: 11; b) a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO: 21, and/or SEQ ID NO: 23; and c) a light chain variable region comprising SEQ ID NO: 31, SEQ I )NO: 33, and/or SEQ ID NO: 35.

[0013] In certain embodiments, the antibodies or antigen binding fragments thereof provided herein comprise:

a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3,and/or SEQ ID NO: 5; and a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9, and/or SEQ ID NO 11; b) a heavy chain variable region comprising SEQ I)NO: 13, SEQ I) NO: 15, and/or SEQ ID NO: 17; and a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO:21, and/or SEQ I) NO: 23; c) a heavy chain variable region comprising SEQ ID NO: 25, SEQ ID NO: 27, and/or SEQ ID NO: 29; and a light chain variable region comprising SEQ ID NO: 31, SEQ

ID NO: 33, and/or SEQ ID NO: 35; or d) a heavy chain variable region comprising SEQ ID NO: 37, SEQ ID NO: 39, and/or SEQ ID NO: 41 and a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO: 21. and/or SEQ ID NO: 23.

[0014] In certain embodiments, the antibodies or antigen binding fragments thereof provided herein comprise a heavy chain variable region selected from the group consisting of: SEQ [D NO: 43, SEQ ID NO: 47, SEQ ID NO: 51 and SEQ ID NO: 55.

[0015] In certain embodiments, the antibodies or antigen binding fragments provided herein comprise a light chain variable region selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 49 and SEQ ID NO: 53.

[00161 In certain embodiments, the antibodies or antigen binding fragments thereof provided herein comprise:

a) a heavy chain variable region comprising SEQ ID NO: 43; and a light chain variable region comprising SEQ ID NO: 45;

b) a heavy chain variable region comprising SEQ ID NO: 47; and a light chain variable region comprising SEQ ID NO: 49;

c) a heavy chain variable region comprising SEQ IDNO: 51; and a light chain variable region comprising SEQ ID NO: 53; or

d) a heavy chain variable region comprising SEQ ID NO: 55; and a light chain variable region comprising SEQ ID NO: 49.

[00171 In certain embodiments, the antibodies provided herein include, for example, 1.4.1, 1.14.4, 1.20.15, and 1.46.11.

100181 In certain embodiments, the antibodies or antigen binding fragments thereof provided herein compete for the same epitope with antibodies 1.4.1, 1.14.4, 1.20.15, and 1.46.11. In certain embodiments, the antibodies or antigen binding fragments thereof provided herein bind to the epitope comprising at least one of the following amino acid residues of PD-Ll: E58, E60, D61, K62, N63 and R13.

100191 In certain embodiments, the antibodies or antigen binding fragments thereof are capable of blocking binding of human PD-L1 to its receptor and thereby providing at least one of the following activities:

a) inducing production of IL-2 in CD4+T cells; b) inducing production ofIFNyin CD4+T cells; c) inducing proliferation of CD4+t-Tcells and d) reversing T reg'ssuppressivefunction.

[0020] In certain embodiments, the antibodies provided herein are a monoclonal antibody, fully human antibody, humanized antibody, chimeric antibody, recombinant antibody, bispecific antibody, labeled antibody, bivalent antibody, or anti-idiotypic antibody.

100211 In certain embodiments, the antigen-binding fragments thereof provided herein are acamelized single domain antibody, a diabody, a scFv, an scFv dimer, a BsFv, a dsFv, a (dsFv)2, a dsFv-dsFv', an Fv fragment, a Fab, a Fab', a F(ab')2, a ds diabody, a nanobody, a domain antibody, or a bivalent domain antibody.

[0022] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin constant region.

[0023] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein, further comprise a conjugate.

[0024] In certain embodiments, the conjugate can be a detectable label, a pharmacokinetic modifying moiety, or a purification moiety.

[0025] In another aspect, the present disclosure provides isolated polynucleotides encoding the antibodies or antigen binding fragments thereof provided herein. In certain embodiments, polynucleotides are provided that encode the amino acid sequences of the antibodies or antigen-binding fragments disclosed herein. in certain other embodiments, vectors are provided that comprise these polynucleotides, and in certain other embodiments, host cells are provided that comprise these vectors. In certain embodiments, methods are provided for expressing one or more of the antibodies or antigen-binding fragments disclosed herein by culturing these host cells under conditions in which the antibodies or antigen-binding fragments encoded by the polynucleotides are expressed from a vector. In certain embodiments, the polynucleotides provided herein are operably associated with a promoter such as a SV40 promoter in a vector. In certain embodiments, host cells comprising the vectors provided herein are Chinese hamster ovary cell, or 293F cell.

[0026] In another aspect, the present disclosure provides kits comprising the antibody or antigen-binding fragment thereof.

[00271 In another aspect, the PD-Li antibodies provided herein, such as the 1.4.1, 1.14.4, 120.15 and 1.46.11 have good tolerability and high in vivo anti-tumor activity in an animal. In certain embodiments, an animal having tumor cells administered with the PD-Li antibodies provided herein has a reduction of the tumor volume by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% as compared to the control animal having similar baseline tumor volume but administered only with vehicle.

[0028] In another aspect, the present disclosure provides methods of detecting presence or level of PD-Li (e.g. human ormonkey) in a biological sample, comprising exposing the biological sample to the antibody or antigen-binding fragment thereof provided herein, and determining the presence or level of the PD-Li in the sample.

[0029] In another aspect, the present disclosure provides methods of identifying an individual having a disorder or condition likely to respond to a PD-LI antagonist, comprising: determining presence or level of PD-LI (e.g. human or monkey) in a test biological sample from the individual with the antibody or antigen-binding fragment thereof provided herein, wherein presence or upregulated level of the PD-Li in the test biological sample indicates likelihood of responsiveness. In certain embodiments, the methods further comprise administering an effective amount of the antibody or antigen-binding fragment thereof provided herein to the individual who has been identified as having a disorder or condition likely to respond to a PD-Li antagonist.

[0030] The present disclosure further provides methods of monitoring therapeutic response or disease progression in a subject treated with a PD-L antagonist, comprising determining presence or level of PD-Li (e.g. human or monkey) in a test biological sample from the individual with the anti-PD-Li antibody or antigen-binding fragment thereof provided herein.

[00311 In another aspect, the present disclosure provides pharmaceutical compositions comprising the antibody or antigen-binding fragment thereof provided herein and one or more pharmaceutically acceptable carriers. In certain of these embodiments, the pharmaceutical carriers may be, for example, diluents, antioxidants, adjuvants, recipients, or non-toxic auxiliary substances.

[0032] In another aspect, the present disclosure provides methods of treating a condition in a subject that would benefit from upregulation of immune response, comprising administering an effective amount of the antibody or antigen-binding fragment thereof provided herein to the subject. In certain embodiments, the subject has upregulated expression of PD-Li.

[0033] Use of the antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a condition that would benefit from upregulation of immune response. In certain embodiments, the condition is cancer or chronic viral infection.

[0033A] In another aspect, the present disclosure provides an antibody or antigen binding fragment thereof provided herein for use in treating a condition that would benefit from upregulation of immune response, preferably wherein the condition is a cancer or a chronic viral infection.

[0033B] In another aspect, the present disclosure provides an antibody or antigen binding fragment thereof provided herein for use in treating an individual having a disorder or a condition likely to respond to a PD-Li antagonist, wherein a presence or level of PD-Li from a test biological sample from the individual indicates likelihood of responsiveness, preferably wherein the condition is a cancer or a chronic viral infection.

[0033C] In another aspect, the present disclosure provides a method of treating an individual having a disorder or a condition likely to respond to a PD-Li antagonist, the method comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof provided herein to the individual, wherein a presence or level of PD-Li from a test biological sample from the individual indicates likelihood of responsiveness, preferably wherein the condition is a cancer or a chronic viral infection.

[0033D] In another aspect, the present disclosure provides use of the antibody or antigen binding fragment thereof provided herein in manufacture of a medicament for treating an individual having a disorder or a condition likely to respond to a PD-Li antagonist, wherein a presence or level of PD-Li from a test biological sample from the individual indicates likelihood of responsiveness, preferably wherein the condition is a cancer or a chronic viral infection.

BRIEF DESCFRIPTION OF FIGURES

[0034] Figure 1 presents the binding of fully human PD-Li antibodies to PD- expressing CHO cell as measured by FACS analysis.

[0035] Figure 2 presents the fully human PD-Li antibodies blocked the binding of PD-i to PD-Li transfected CHO cells as measured by FACS analysis.

7A

[0036] Figure 3 shows that the fully human PD-Li antibodies specifically bound to PD-Li, but did not bind to PD-L2, as measured by FACS analysis.

[0037] Figure 4 shows that the fully human PD-Li antibodies bound to human and cynomolgus monkey PD-LI.

[0038] Figure 5 is the full kinetics of binding affinity of PD-L antibodies to human PD-Li ranging from 2.26E-10 to 4.78E-10 mol/L as determined by surface plasmon resonance.

[0039] Figure 6 illustrates the effect of fully human anti-PD-Li antibodies on IFNy production in specific T cell response.

[0040] Figure 7 shows that fully human anti-PD-Li antibodies enhanced specific T cell proliferation.

[0041] Figure 8 shows that fully human PD-Li antibodies enhanced IFNy production in mixed lymphocyte reaction (MLR).

[0042] Figure 9 illustrates the effect of fully human anti-PD-Li antibodies on IL-2 production in MLR.

[0043] Figure 10 shows that anti-PD-Li antibodies promoted T cell proliferation in MLR.

[0044] Figure I Ishows that anti-PD-LI antibodies reversed Treg's suppressive function.

[0045] Figure 12 shows the anti-PD-Li antibodies lacked ADCC on activated T cells.

[0046] Figure 13 shows the anti-PD-Li antibodies lacked CDC on activated T cells.

[00471 Figure 14A and 14B show cross-reactivity of anti-PD-LI antibodies with human/mouse PD-1. 2 ig/ml of 1.14.4 antibody was coated at 96-well plate overnight and incubated with (Figure 14A) hPD-LI-His protein and (Figure 14B) mPD-LI-His protein, then HRP-anti-His antibody were added for detection.

[0048] Figure 15 shows hot spot residues mapped on hPD-L1 structure. Binding site of antibody 1.14.4. Data were from table 3. Colors on the pictures are to help distinguish the differences between epitopes.

100491 Figure 16 shows good in vivo tolerability of the hPD-LI antibody 1.14.4. Three doses of antibody 1.14.4 (3mg/kg, 10mg/kg and30mg/kg) were administrated via multi intraperitoneal injections to the humanized B-hPD-1 mouse. No significant change of the body weight was observed during the experiment.

[0050] Figure 17 shows significant in vivo inhibition of the hPD-Li antibody 1.14.4 to the tumor cell growth. After 19 days of antibody administration, all the three doses of antibody 1.14.4 (3mg/kg, 10mg/kg and30mg/kg) showed significant anti-tumor effects indicated by tumor growth inhibitions (TGI) of >40%.

DETAILED DESCRIPTION OFTHE INVENTION

[0051] The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be nade without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety.

100521 Definitions

[0053] The term "antibody" as used herein includes anyimmunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific bivalentt) antibody that binds to a specific antigen. A native intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as , 6,, ,i, and, and mammalian light chains are classified as ?, or K. The antibody has a "Y" shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variables region in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light (L) chain CDRs including LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRs including HCDR, HCDR2, HCDR3). CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A. M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, A.M., J.MolBiol., 196,901 (1987); Chothia, C. et aL, Nature. Dec 21 28;342(6252):877-83 (1989) ; Kabat E.A. et al.. National Institutes of Health. Bethesda, Md. (1991)). The three CDRs are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of a, 6, s, y, and p heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgGI (f1 heavy chain), IgG2 (f2 heavy chain), IgG3 (3 heavy chain), IgG4 (y4 heavy chain), IgA1 (al heavy chain), or IgA2 (u2 heavy chain).

[0054] The term "antigen-binding fragment" as used herein refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer bivalentt diabody), a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

[0055] "Fab" with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond.

[0056] "Fab" refers to a Fab fragment that includes a portion of the hinge region.

[00571 "F(ab')2"refers to a dimer of Fab'.

[0058] "Fc" with regard to an antibody refers to that portion of the antibody consisting of the second and third constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bonding. The Fe portion of the antibody is responsible for various effector functions such as ADCC, and CDC, but does not function in antigen binding.

[0059] "Fv" with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0060] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879(1988)).

[0061] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of a scFv connected to the Feregion of an antibody.

[0062] "Camelized single domain antibody," "heavy chain antibody," or "HCAb" refers to an antibody that contains two VH domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231(1-2):25-38 (1999); Muyidermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94/04678; W094/25591: U.S. Patent No. 6,005,079). Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas). Although devoid of light chains, camelized antibodies have an authentic antigen-binding repertoire (Hamers-Casterman C. et al, Nature. Jun 3;363(6428):446-8 (1993); Nguyen VK. etal. "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," Immunogenetics. Apr;54(1):39-47 (2002); Nguyen VK. et al.Immunology. May;109(1):93-101 (2003)). The variable domain of a heavy chain antibody

(VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASE3 J. Nov;21(13):3490-8. Epub 2007 Jun 15 (2007)).

[0063] A "nanobody" refers to an antibody fragment that consists of a VHM domain from a heavy chain antibody and two constant domains, CH2 and CH3.

[0064] "Diabodies" include small antibody fragments with two antigen-binding sites, wherein the fragments comprise a VH domain connected to a VL domain in the same polypeptide chain (VH-VL or VH-VL) (see, e.g, Holliger P. et al., Proc Natl Acad Sci U S A. Jul 15;90(14):6444-8 (1993);EP404097;W093/11161). Byusing a linkerthat is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites. The antigen-binding sites may target the same of different antigens (or epitopes).

100651 A "domain antibody" refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. In certain instances, two or more VH domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody. The two V domains of a bivalent domain antibody may target the same or different antigens.

100661 In certain embodiments, a "(dsFv) 2 " comprises three peptide chains: two VH moieties linked by a peptide linker and bound by disulfide bridges to two VL moieties.

[0067] In certain embodiments, a "bispecific ds diabody" comprises VH1-L2 (linked by a peptide linker) bound to VL-VI12 (also linked by a peptide linker) via a disulfide bridge between V1w and VL1.

[0068] In certain embodiments, a "bispecific dsFv" or dsFv-dsFv" comprises three peptide chains: a VH-H2 moiety wherein the heavychains are linked by a peptide linker (e.g., a long flexible linker) and bound to Vu and VL2 moieties, respectively, via disulfide bridges, wherein each disulfide paired heavy and light chain has a different antigen specificity.

[0069] In certain embodiments, an "scFv dimer" is a bivalent diabody or bivalent ScFv (BsFv) Comprising VH-VL (linked by a peptide linker) dimeried with another VH-VL moiety such that VH'S of one moiety coordinate with the VL'S of theother moiety and form two binding sites which can target the same antigens (or eptipoes) or different antigens (or eptipoes). In other embodiments, an "scFv dimer" is a bispecific diabody comprising Vm VuL2 (linked by a peptide linker) associated with Vu-VH2 (also linked by a peptide linkeT) such that Vm and VL coordinate and Vm and Vu coordinate and each coordinated pair has a different antigen specificity.

[0070] The term "fully human" as used herein, with reference to antibody or antigen binding fragment, means that the antibody or the antigen-binding fragment has or consists of amino acid sequence(s) corresponding to that of an antibody produced by a human or a human immune cell, or derived from a non-human source such as a transgenic non-human animal that utilizes human antibody repertoires or other human antibody-encoding sequences. In certain embodiments, a fully human antibody does not comprise amino acid residues (in particular antigen-binding residues) derived from a non-human antibody.

[0071] The term "humanized" as used herein, with reference to antibody or antigen binding fragment, means that the antibody or the antigen-binding fragment comprises CDRs derived from non-human animals, FR regions derived from human, and when applicable, the constant regions derived from human. A humanized antibody or antigen-binding fragment is useful as human therapeutics in certain embodiments because it has reduced immunogenicity in human. In some embodiments, the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster. In some embodiments, the humanized antibody or antigen-binding fragment is composed of substantially all human sequences except for the CDR sequences which are non-human. In some embodiments, the FR regions derived from human may comprise the same amino acid sequence as the human antibody from which it is derived, or it may comprise some amino acid changes, for example, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, orI changes of amino acid. In some embodiments, such change in amino acid could be present in heavy chain FR regions only, in light chain FR regions only, or in both chains. In some preferable embodiments, the humanized antibodies comprise human FRI-3 and human JH and J1 .

[0072] The term "chimeric" as used herein, means an antibody or antigen-binding fragment, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species. In an illustrative example, a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human species, such as from mouse.

[0073] "PD-L1" as used herein refers to programmed cell death ligand I (PD-L1, see, for example, Freeman et al. (2000)J. Exp. Med 192:1027). Representative amino acid sequence of human PD-Li is disclosed under the NCBI accession number: NP_054862.1, and the representative nucleic acid sequence encoding the human PD-Li is shown under the NCBI accession number: NM_014143.3. PD-L1 is expressed in placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found on many tumor or cancer cells. PD-LIbinds to its receptor PD-i or 137-1, which is expressed on activated T cells, B cells and nyeloid cells. The binding of PD-LI and its receptor induces signal transduction to suppress TCR-mediated activation of cytokine production and T cell proliferation. Accordingly, PD-L1 plays a major role in suppressing immune system during particular events such as pregnancy, autoimmune diseases, tissue allografts, and is believed to allow tumor or cancer cells to circumvent the immunological checkpoint and evade the immune response.

[0074] "Anti-PD-1 antibody" as used herein refers to an antibody that is capable of specific binding to PD-Li (e.g. human or monkey PD-Li) with an affinity which is sufficient to provide for diagnostic and/or therapeutic use.

[0075] The term "specific binding" or "specifically binds" as used herein refers to a non random binding reaction between two molecules, such as for example between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically bind human and/or monkey PD-L1 with a binding affinity (KD) of $10YMI(e.g., <5x10- 7 M 2x10- 7 NM i04 M, 5x10 M<x10'M, <10 M, 5x10 9 M, 52x10 MI, <10- M, about I0°' M, 10-° M to 10- M, 10-1 M to 105 M, or 010 M to 10 M). KD as used herein refers to the ratio of the dissociation rate to the association rate (konf/ko), may be determined using surface plasmon resonance methods for example using instrument such as Biacore.

[0076] The ability to "block binding" or "compete for the same epitope" as used herein refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g. human PD-Li and an anti-PD-LI antibody) to any detectable degree. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 50%. In certain embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0077] The term "epitope" as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies may bind the same epitope within an antigen if they exhibit competitive binding for the antigen. For example, if an antibody or antigen-binding fragment as disclosed herein blocks binding of the exemplary antibodies such as 1.4.1, 1.14.4, 1.20.15, and 1.46.11 to human PD-Li, then the antibody or antigen-binding fragment may be considered to bind the same epitope as those exemplary antibodies.

[0078] A particular amino acid residue within the epitope can be mutated, e.g. by alanine scanning mutagenesis, and mutations that reduce or prevent protein binding are identified. An "alanine scanning mutagenesis" is a method that can be performed for identifying certain residues or regions of a protein that affect the interaction of the epitope with another compound or protein that binds to it. A residue or group of target residues within the protein is replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine, or a conservative amino acid substitution). Any mutation of the amino acid residues or codons encoding the same that reduces binding of the protein more than a threshold or reduces binding of the protein to the maximal degree than other mutations is likely to be within the epitope bound by the protein. In certain embodiments of the present disclosure, the epitope that is critical for the PD-Li antibody comprises at least one of the amino acid residues of E58, E60, D61, K62, N63 and RI13.

[0079] "14.1" as used herein refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 43, light chain variable region of SEQ ID NO: 45, and a human constant region of IgG4 isotype.

[0080] "1.14.4" as used herein refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 47, light chain variable region of SEQ ID NO: 49, and a human constant region of IgG4 isotype.

100811 "1.20.15" as used herein refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 51, light chain variable region of SEQ ID NO: 53, and a human constant region of IgG4 isotype.

[0082] "1.46.11" as used herein refers to a fully human monoclonal antibody having a heavy chain variable region of SEQ ID NO: 55, light chain variable region of SEQ ID NO: 49, and a human constant region of IgG4 isotope.

[0083] A "conservative substitution" with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g. Met, Ala, Val, Leu, and lie), amongresidues with neutral hydrophilic side chains (e.g. Cys, Ser, Thr, Asn andGln), among residues with acidic side chains (e.g. Asp, Glu), among amino acids with basic side chains (e.g. His, Lys, and Arg), or among residues with aromatic side chains (e.g. Trp, Tyr, and Phe). As known in the art, conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.

[00841 "Percent (%) sequence identity" with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids). Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI), see also, Altschul S.F. et al, J. Mol. Biol., 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of European Bioinformatics Institute, see also, Higgins D.G. et al, Methods in Enzymology, 266:383-402 (1996); Larkin MA et al, Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or may customize the parameters as appropriate for the alignment, such as for example, by selecting a suitable algorithm.

[0085] "T cell" as used herein includes CD4' T cells, CD8* T cells, T helper I type T cells, Thelper 2 type T cells, T helper 17 typeT cells and inhibitoryT cells.

[0086] "Effector functions" as used herein refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as C complex and Fc receptor. Exemplary effector functions include: complement dependent cytotoxicity (CDC) induced by interaction of antibodies and Clg on the C complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by binding of Fe region of an antibody to Fe receptor on an effector cell; and phagocytosis.

[0087] "Cancer" or "cancerous condition" as used herein refers to any medical condition mediated by neoplastic or malignant cell growth, proliferation, or metastasis, and includes both solid cancers and non-solid cancers such as leukemia. "Tumor" as used herein refers to a solid mass of neoplastic and/or malignant cells.

[0088] "Treating" or "treatment" of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof With regard to cancer, "treating" or "treatment" may refer to inhibiting or slowing neoplastic or malignant cell growth, proliferation, or metastasis, preventing or delaying the development of neoplastic or malignant cell growth, proliferation, or metastasis, or some combination thereof With regard to a tumor, "treating" or "treatment" includes eradicating all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

[0089] An "isolated" substance has been altered by the hand of man from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide is "isolated" if it has been sufficiently separated from the coexisting materials of its natural state so as to exist in a substantially pure state. In certain embodiments, the antibodies and antigen-binding fragments have a purity of at least 90%, 93%, 95%, 96%, 97%, 98%, 99% as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatographic methods (such as ion exchange chromatography or reverse phase H1PLC).

[0090] The term "vector" as used herein refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein. A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (eg., SV40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating.

[00911 The phrase "host cell" as used herein refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced.

100921 A "disease associated with or related to PD-L" as used herein refers to any condition that is caused by, exacerbated by, or otherwise linked to increased or decreased expression or activities of PD-L1 (e.g. a human PD-L).

[0093] The term therapeuticallyy effective amount" or "effective dosage" as used herein refers to the dosage or concentration of a drug effective to treat a disease or condition associated with human PD-Li. For example, with regard to the use of the antibodies or antigen-binding fragments disclosed herein to treat cancer, a therapeutically effective amount is the dosage or concentration of the antibody or antigen-binding fragment capable of eradicating all or part of a tumor, inhibiting or slowing tumor growth, inhibiting growth or proliferation of cells mediating a cancerous condition, inhibiting tumor cell metastasis, ameliorating any symptom or marker associated with a tumor or cancerous condition, preventing or delaying the development of a tumor or cancerous condition, or some combination thereof.

[0094] The term "pharmaceutically acceptable" indicates that the designated carrier, vehicle, diluent, excipient(s), and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof

[0095] Anti-PD-L1 antibody

[00961 In one aspect, the present disclosure provides anti-PD-LI antibodies and the antigen-binding fragments thereof PD-1, also called as CD279, is known as a key immune checkpoint receptor expressed by activated T cells, which mediates immunosuppression. PD 1 ligand I (PD-Li) is a 40 kDa transmembrane protein expressed on various tumor cells, stromal cells or both, and binds to PD-1. Inhibition of the interaction between PD-1 and PD L1 can enhance T-cell responses and thus mediates anti-cancer activity.

[00971 In certain embodiments, the present disclosure provides exemplary fully human monoclonal antibodies 14.1, 1 14.4, 120.15, and 1.46. 11, whose CDR sequences are shown in the below Table 1, and heavy or light chain variable region sequences are also shown below.

[0098] Table 1

CDR1 CDR2 CDR3

SEQ ID NO: I SEQ ID NO: 3 SEQ ID NO: 5

1.4.11IRTYYWG Y1YYSGSTRYN1SLKS LSYFFDY VH(3051 1) SEQ ID NO: 2 SEQ ID NO: 4 SEQ ID NO: 6

ATT AGA ACT TAT ATC TAT TAT AGT GGG AGC ACC CGC CTT AGC TAC TTC TAC TAC TGG 'AC AAC CCG TCC CTC TTTGACTAC G C AAG AGT SEQ ID NO: 7 SEQ ID NO: 9 SEQ ID NO: 11

L(30027) SGDKIGDKYAC QDTKRPS QAWDSGTVI

SEQ ID NO: 8 SEQ ID NO: 10 SEQ ID NO: 12 TCT GGA CAT CGGGG A AAA TTG GGG CAA GAT ACC AAG AGC GG AC GAT AAA TAT CGG CCC TCA ATA GCTTGC

SEQ ID NO: 13 SEQ H) NO: 15 SEQ ID NO: 17

1.14.4- SYAMS GISGSGCFTYYAI)SVK(I PPRGYNYGPF)Y

SEQ ID NO: 14 SEQ ID) NO: 16 SEQ ID NO: 18 GGT ATT AGT GGT CCT CCT CGT GGA AGC TAT GCC AGT GCT GGT TTC ACT T ATG AGT TAC TAC GCA GAC T T G:AACJ TAGC TCC GTG AAG GGC CCTTTTGACTAC

SEQ ID NO: 19 SEQ I )NO: 21 SEQ ID NO: 23

2841) GGNNIGSKSVH DDSDRPS QVWDSSSDI-IVV

SEQ ID NO: 20 SEQ ID NO: 22 SEQ ID NO: 24 GGG GGA AAC CGGGrii&v AAC I ATT GA CAT GAT AGC GAC AGGTGTGAT AGT AAA AGT CGG CCC TCA CAC TG GTA GTA CAC

SEQ ID NO: 25 SEQ ID NO: 27 SEQ ID NO: 29 1.20.15- SISNYWG SIYYSGST'NYNPPLKS LTYYFDY VH-(307112)

SEQ ID NO: 26 SEQ ID NO: 28 SEQ ID NO: 30

AGT ATT AGT AGT ATC TAT TAT AGT ATATAGG GGG AGC ACG AAC CTG ACC TAC TAC AAC TAC T GG T AC AATCCG CCC CTC TTTGATTAC GC AAG AGT

SEQ ID NO: 31 SEQ ID NO: 33 SEQ ID NO: 35 1.20.15- SGDKLGDKYAC QDSKRPS QTWDSSTVV VL(29907) SEQ ID NO: 32 SEQ ID NO: 34 SEQ ID NO: 36 TCT GGA GAT CAGACGTGGGAC AAA TTG GGG CAA GAT AGC AAG AGC AGC ACTGTG GAT AAA TAT CGG CCCTCA GTA GCTTGC SEQ ID NO: 37 SEQ ID NO: 39 SEQ ID NO: 41 1.46.11- SYAMS GFSGSGFITYYADSVKG P1RGYNYGPFDY VH(30626) SEQ ID NO: 38 SEQ ID NO: 40 SEQ ID NO: 42 GGT TTT AGT GGT AGT CCT CCT CGT GGA AGC TAT GCC GGT TTTATTACATAC ATGAT TAC GCA GAC TC TAC AACTAT GGC ATG AGT TAC r GCA k GACI T CCT TTT GAC TAC GTG AAG GGC SEQ ID NO: 19 SEQ ID NO: 21 SEQ ID NO:223 1.46.11- GGNNIGSKSVH DDSDRPS QVWDSSSDHVV VL(29841) SQ SEQ ID NO: 20 SEQ ID NO: 22 SEQ ID NO: 24 GGG GGA AAC CAGGTGTGGGAT AAC ATT GGA GAT GAT AGC GAC AGT AGTAGT GAT AGT AAA AGT CGG CCC TCA C GTA CAC CAC GIG GTA

[0099] 1.4.1-V(30511): (SEQ ID NO:43 for amino acid and SEQ ID NO:44 fornucleic acid) with heavy chain CDRs 1-3: SEQ ID NOs: 1, 3, 5 are amino acid sequences and SEQ ID NO:2, 4, 6 are nucleic acid sequences, respectively:

V segment: IGHV4-39*01 D segrnent: IGHD1 -26*01

J segment: IGHJ4*02

Q L Q L C E S G P G- L V K [P S 1 CAG CTG CAA CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG CCT TCG

E S L S L T C T V S G G S I S 46 GAG TCC CTG TCC CTC ACC TGC ACT GTC TCT G GC TCC ATC AGC

CDR1

I R T Y Y W G W I R C P P G T 91 ATT AGA ACT TAC TAC TGG GGC TGG ATC CGC CAG CCC CCA GGG AC

CDR2

G L E W M G Y I Y Y S G S T R 136 GGG CTG GAG TGG ATG GGG TAT ATC TAT TAT AGT GGG AGC ACC CGC

CDR2

Y N P S L K S R V T I S V D T 181 TAC AAC CCG TCC CTC AAG AGT CGA GTC ACC ATA TCC GTA GAC AC

S K N Q F S L K L S S V T A A 226 TCC AAG AAC CAG TTC TCC CTG AAG CTG AGC TCT GTG ACC GCC GCA

CDR3

D T A V Y Y C A R L S Y F F D 271 GAC ACG GCT GTG TAT TAC TGT GCG AGA CTT AGC TAC TTC TTT GAC

CDR3

Y W G C G T L V T V S S (SEQ ID NO:43) 316 TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA (SEQ ID NO:44)

[001001 1.4.1-VL(30027): (SEQ IDNO:45 for amino acid and SEQ ID NO:46 for nucleic acid) with light chain CDRs1-3: SEQ iD NOs: 79, 11 are amino acid sequences and SEQ ID NO8, 10, 12 are nucleic acid sequences, respectively:

V segment: IGLV3-1*01 J segment: IGLJ2*01

S Y E L T Q P P S V S V S P G 1 TCC TAT GAA CTG ACT CAG CCA CCC TCA GTG TCC GTG TCC CCA GGA

CDRI

Q T A S I[ T C S G D K L G D K 46 CAG ACA GCC AGC AITC ACC TGC TCT GGA GAT AAA TTG GGG GAT AAA

CDRI

Y A C W Y Q Q K P G Q S P V M 91 TAT GCT TGC TGG TAT CAG CAG AAG CCA GGC CAG TCC CCT GTG ATG

CDR2

V I Y Q D T K R P S G I P E R 136 GTC ATC TAT CAA GAT ACC AAG CGG CCC TCA GGG ATC CCT GAG CGA

F S G S N S G N T A T L T I S 181 TTC TCT GGC TCC AAC TCT GGG AAC ACA GCC ACT CTG ACC ATC AGC

CDR3

G T L A M D E A D Y Y C Q A W 226 GGG ACC CTG GCT ATG GAT GAG GCT GAC TAT TAT TGT CAG GCG TGG

CDR3

D S G T V I F G G G T K L T V 271 GAC AGC GGC ACT GITG ATA TTC GGC GAGG G ACC AAG CTG ACC GTC

(SEQ ID N0:45) 316 CTA (SEQ ID NO:46)

[001011 1.14.4-VH(29812): (SEQ ID NO:47 for amino acid and SEQ ID NO:48 for nucleic acid) with heavy chain CDR-s 1-3: SEQ ID NOs: 13, 15, 17 are amino acid sequences and SEQ ID NO:14, 16, 18 are nucleic acid sequences, respectively:

V segment: IGHV3-23*01 D segment: IGHD5-5*01 J segment: IGHJ4*02

V Q L L E S G G G L V Q P G

1 GAG GTG CAA CTG TTG GAG TCT GGG GGA GGC TTG GTA CAG CCT GGG

G S L R L S C A A S G F T F S 46 GGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACC TT AGC

CDR1

S Y A M S W V R A P G K G L 91 AGC TAT GCC ATG AGT TGG GTC CGC CAG GCT CCA GGG AAG GGG CT

CDR2

E W V S G I S G S G G F T Y Y 136 GAG TGG GTC TCA GGT ATT AGT GGT AGT GGT GGT TTC ACT TAC TAC

CDR2

A D S V K G R F T I S R D N S 181 GCA GAC TCC GTG AAG GGC CGG TTC ACC ATC TCC AGA GAC AAT TCC

K N T L Y L Q M N S L R A E

) 226 AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAG GAC

CDR3

T A V Y Y C A K P P R G Y N Y 271 AC GCC GTA TAT TAC TGT GCG AAA CCT CCT CGT GGA TAC AAC TAT

CDR3

G P F D Y W G Q C T L V T V S 316 GGC CCT TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC

S (SEQ ID NO:47) 361 TCA (SEQ ID NO:48)

1001021 1.14.4-VL and 1.46.11-VL (29841): (SEQ ID NO:49 for amino acid and SEQ ID NO:50 for nucleic acid) with light chain CDR-s 1-3: SEQ ID NOs: 19, 21, 23 are amino acid sequences and SEQ 1D NO:20, 22, 24 are nucleic acid sequences, respectively:

V segment: IGLV3-21*02 J segment: IGLJ2*01

S Y V L T Q P P S V S V A P G

1 TCC TAT GTG CTG ACT CAG CCA CCC TCG GTG TCA GTG GCC CCA GGA

CDRI

TT A R I T C G G N N I G S K 46 CAG ACG GCC AGG ATT ACC TGT GGG GGA AAC AAC ATT GGA AGT AAA

CDR!

S V H W Y Q Q K P G Q A P V L 91 AGT GTA CAC TGG TAC CAG CAG AAG CCA GGC CAG GCC CCT GTG CTG

CDR2

V V Y D D S D R P S G I P E R 136 GTC GTC TAT GAT GAT AGC GAC CGG CCC TCA GGG ATC CCT GAG CGA

F S G S N S G N T A T L T I S 181 TTC TCT GGC TCC AAC TCT GGG AAC ACG GCC ACC CTG ACC ATC AGC

CDR3

R V E A G D E A D Y Y C Q V W 226 AGG GTC GAA GCC GGG GAT GAG GCC GAC TAT TAC TGT CAG GTG TGG

CDR3

D S S S D H V V F G G G T K L 271 GAT AGT AGT AGT GAT CAC GTG GTA TTC GGC GGA GGG ACC AAG CTG

T V L (SEQ ID NO:49) 316 ACC GTC CTA (SEQ ID N:50)

[001031 1.2015-VI(30712): (SEQ ID NO51 Or amino acid and SEQ ID NO:52 for nucleic acid) with heavy chain CDRs 1-3: SEQ ID NOs: 25, 27, 29 are amino acid sequences and SEQ ID NO:26, 28, 30 are nucleic acid sequences, respectively:

Segment: IGHV4-39*01 D segment: undetermined J segment: IGHJ4*02

Q L Q L E S G P G L V K P S

1 CAG CTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG CCT TCG

E T L S L T C T V S G G S I S 46 GAG ACC CTG TCC CTC ACC TGC ACT GTC TCT GGT GGC TCC ATC AGC

CDRI

S I S N Y W G W I R Q P P G K 91 AGT ATT AGT AAC TAC TGG GGC TGG ATC CGC CAG CCC CCA GGG AAG

CDR2

G L E W I G S I Y Y S G S T N 136 GGG CTG GAGT AT AGT ATC TAT TAT AGT GGG AGC ACG AAC

CDR2

Y N P P L K S R V T I S V D T 181 TAC AAT CCG CCC CTC AAG AGT CGA GTC ACC ATA TCC GTA GAC ACG

T K N Q F S L K L S S V T A A 226 ACC AAG AAC CAG TTC TCC CTG AAG CTG AGC TCT GTG ACC GCC GCA

CDR3

D T A V Y Y C A R L T Y Y F D 271 GCACACG GCT GTG TAT TAC TGT GCG AGA CTG ACC TAC TAC TTT GAT

CDR3

Y W G GC M L V T V S S (SEQ ID NO:51) 316 TAC TGG GGC CAG GGA ATG CTG GTC ACC GTC TCC TCA (SEQ ID NO:52)

[001041.2015-VL(29907): (SEQ IDNO:53 for amino acid and SEQ IDNO:54 fornucleic acid) with light chain CDRs 1-3: SEQ ID NOs: 31, 33, 35 are amino acid sequences and SEQ ID NO:32, 34, 36 are nucleic acid sequences, respectively:

V segment: IGLV3-1*01 J segment: IGLJ2*01

S Y D L T 9 P P S V S V S [P G 1 TCC TAT GAC CTG ACT CAG CCA CCC TCA GTG TCC GTC TCC CCA GGA

CDR1

T A S I T C S G ) K L G ) K 46 CAG ACA GCC AGC ATC ACC TGC TCT GAGAT AAA TG GGG GAT AAA

CD)R1

Y A C W Y Q Q K [) G S ) L L 91 TAT GCT TGC TGG TAT CAG CAG AAG CCA GGC CAG TCC CCT ITG CTG

CDR2

V Q IQ D S K R P S G I P A R 136 GCATAC CAG CAA GAT AGC AAG CGG CCC TCA GGG ATC CCT GCG CGA

F S G S N S G N T A T L T I S 181 TTC TCT GGC TCC AAC TT GGG AAC ACA GCC ACT CTG ACC ATC AGC

CDR3

G T 9 A VI D E A ) Y F C Q T W 226 GGG ACC CAG GCT ATG GAT GAG GCT GAC TAT TTC TGT CAG ACG TGG

CDR3

D S S T V V F G G G T K L T V 271 GAC AGC AGC ACT GTG GTA TTC GGC GGA GGG ACC AAG CTG ACC GTC

(SfEQ D NO:53) 316 CTA (SEQ ID NO:54)

[001051 1.46.11-VH(30626): (SEQ D NO:55 for amino acid and SEQ ID NO:56 for nucleic acid) with heavy chain CDR-s 1-3: SEQ ID NOs: 37, 39, 41 are amino acid sequences and SEQ ID NO:38, 40, 42 are nucleic acid sequences, respectively:

V segment: IGHV3-23*01 D segment: IGHD5-5*0I J segment: IGIJ4*02

E V 9 L L E S G G G L V 9 [ G 1 GAG GTG CAG TTG TTG GAG CT GGG GGA GGC TTG GIA CAG CCT GGG

C S L R L S C A A S G F T F S 46 G TCC CTG AGA CTC TCC TGT GCA GCC TCT G TTC ACC TTT AGC

CDRI

S Y A M S W V R Q A P G K G L 91 AGC TAT GCC ATG AGT TGG GTC CGC CAG GCT CCA GGG AAG GGG CTG

CDR2

E W V S G F S G S G F I T Y Y 136 GAG TGG GTC TCA GGT TTT AGT GGT AGT GGT TTT ATT ACA TAC TAC

CDR2

A 1) S V K G R F T I S R D N S 181 GCA GAC TCC GTG AAG GGC CGG TTC ACC ATC TCC AGA GAC AAT TCC

K N TI L Y L Q M N S L R A F D 226 AAG AAT ACG CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAG GAC

CDR3

T A V Y Y C A M P P R G Y N Y 271 ACG GCC GTA TAT TAC TGT GCG ATG CCT CCT CGT GGA TAC AAC TAT

CDR3

G P F 1) Y W G Q G T L V I V S 316 GGC CCT TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC

S (SEQ ID NO:55) 361 TCA (SEQ [ID NO:56)

[001061 1.46.11-VL(29841):(SEQ ID NO:49 for amino acid and SEQ ID NO:50 for nucleic

acid) with light chain CDRs 1-3: SEQ ID NOs: 19, 21,23 are amino acid sequences and SEQ ID NO:20, 22, 24 are nucleic acid sequences, respectively:

V segment: IGLV3-21*02 J segment: IGLJ2*01

S Y V L T Q P P S V S V A P C I TCC TAT GTG CTG ACT CAG CCA CCC TCG GTG TCA GTG GCC CCA GGA CDRI

Q T A R I T C G G N N I G S K 46 CAG ACG GCC AGG ATT ACC TGT GGG GGA AAC AAC ATT GGA AGT AAA

CDR1

CDR2

V V Y D D S D R P S G I P E R 136 GTC GTC TAT GAT GAT AGC GAC CGGCCC TCA GGG ATC CCT GAG CGA

F S G S N S G N T A T L T I S 181 TTCTCCT GGC TCC AAC TCT GGG AAC ACG GCC ACC CTG ACC ATC AGC

CDR3

T V L (SEQ ID NO:49) 316 ACC GTC CTA (SEQ ID NO:50)

[001071 In some embodiments, the anti-PD-Li antibodies and the antigen-binding fragments thereof comprise a heavy chain CDR sequences selected from the group consisting of: SEQ ID NOs: 1, 3, 5, 13, 15, 17, 25, 27, 29, 37, 39 and 41. In some embodiments, the anti-PD-Li antibodies and the antigen-binding fragments thereof comprise a light chain CDR sequences selected fromthe group consisting of: SEQ ID NOs: 7, 9, 11, 19, 21, 23, 31, 33, and 35.

[001081 In some embodiments, the anti-PD--1 antibodies and the antigen-binding fragments thereof comprise a heavy chain variable region selected from the group consisting of: a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5; a heavy chain variable region comprising SEQ 1D NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 17; a heavy chain variable region comprising SEQ ID NO: 25, SEQ ID NO: 27, and/or SEQ

ID NO: 29; and a heavy chain variable region comprising SEQ ID NO: 37, SEQ ID NO: 39, and/or SEQ ID NO: 41.

[001091 In some embodiments, the anti-PD-L1 antibodies and the antigen-binding fragments thereof comprise a light chain variable region selected from the group consisting of: a light chain variable region comprising SEQ ID NO: 7, SEQ ID NO: 9, and/or SEQ 1D NO: 11; a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO: 21, and/or SEQ ID NO: 23; and a light chain variable region comprising SEQ 11) NO: 31, SEQ 11) NO: 33, and/or SEQ ID NO: 35.

[001101 In some embodiments, the anti-PD-L Iantibodies and the antigen-binding fragments thereof comprising: a) a heavy chain variable region comprising SEQ ID NO: 1, SEQ ID NO: 3, and/or SEQ ID NO: 5; and a light chain variable region comprising SEQ ID NO: 7, SEQ 11) NO: 9, and/or SEQ ID NO: 11; b) a heavy chain variable region comprising SEQ I) NO: 13, SEQ ID NO: 15, and/or SEQ ID NO: 17; and a light chain variable region comprising SEQ ID NO: 19, SEQ I) NO: 21, and/or SEQ ID NO: 23; c)a heavy chain variable region comprising SEQ ID NO: 25, SEQ ID NO: 27, and/or SEQ ID NO: 29; and a light chain variable region comprising SEQ I)NO: 31, SEQ ID NO: 33, and/or SEQ I)NO: 35; or d) a heavy chain variable region comprising SEQ ID NO: 37, SEQ ID NO: 39, and/or SEQ ID NO: 41 and a light chain variable region comprising SEQ ID NO: 19, SEQ ID NO: 21, and/or SEQ ID NO: 23.

[001111 A skilled artisan will understand that the CDR sequences provided in Table I can be modified to contain one or more substitutions of amino acids, so as to provide for an improved biological activity such as improved binding affinity to human PD-L1. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed with phage display technology, and then screened for the binding affinity to human PD-LI. For another example, computer software can be used to virtually simulate the binding of the antibodies to human PD-Li, and identify the amino acid residues on the antibodies which fori the binding interface. Such residues may be either avoided in the substitution so as to prevent reduction in binding affinity, or targeted for substitution to provide for a stronger binding. In certain embodiments, at least one (or all) of the substitution(s) in the CDR sequences is conservative substitution.

1001121 In certain embodiments, the antibodies and the antigen-binding fragments thereof comprise one or more CDR sequences having at least 80% (e.g. at least 85%, 88%, 90%,

91% 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) listed in Table 1, and in the meantime retain the binding affinity to human PD-L1 at a level similar to or even higher than its parental antibody having substantially the same sequence except that the corresponding CDR sequence is in 100% sequence identity to that (or those) listed in Table 1.

[001131 In certain embodiments, the anti-PD-Li antibodies and the anti fragments thereof are fully human. The fully human antibodies do not have the issues of immunogenicity in human and/or reduced binding affinity as often observed with humanized antibodies.

[001141 In some embodiments, the fully human anti-PD-L1 antibodies and the antigen binding fragments thereof comprise a heavy chain variable region selected from the group consisting of: SEQID) NO: 43, SEQ I )NO: 47, SEQ I )NO: 51, SEQ I) NO: 55, and a homologous sequence thereof having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity; and/or a light chain variable region selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, and a homologous sequence thereof having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity. Theses fully human antibodies retain the binding affinity to human PD-L, preferably at a level similar to one of the exemplary antibodies: 1.4.1, 1.14.4, 1.20.15, and 1.46.11.

[001151 In some embodiments, the fully human anti-PD-Li antibodies and the antigen binding fragments thereof comprise: a) a heavy chain variable region comprising SEQ I) NO: 43; and a light chain variable region comprising SEQ ID NO: 45; b) a heavy chain variable region comprising SEQ ID NO: 47; and a light chain variable region comprising SEQ ID NO: 49; c) a heavy chain variable region comprising SEQ ID NO: 51; and a light chain variable region compiising SEQ ID NO: 53; or d) a heavy chain variable region comprising SEQ I) NO: 55; and a light chain variable region comprising SEQ ID NO: 49.

[001161 Also contemplated herein are antibodies and the antigen-binding fragments that compete for the same epitope with the anti-PD-L1 antibodies and the antigen-binding fragments thereof provided herein. In certain embodiments, the antibodies block binding of 14.1, 1.14.4, 1.20.15, and 1.46.11 to human or monkey PD-Li, for example, at anIC5 0 value (i.e.50% inhibition concentration) of below 10`M, below 10-7 M, below 10-5 M below 10 M, below 10- M, below 10' M, or belowI04° M. The IC values are determined based on a competition assay such as ELISA assays, radioligand competition binding assays, and FACS analysis.

[00117] In some embodiments, the anti-PD-L1 antibodies and the antigen-binding fragments thereof provided herein are capable of specifically binding to human PD-LI with a binding affinity (Kd) of <10- M (e.g., <5x10 4 M 2X10-M$<10 M, 5x10 8 M, 2x10~ M, 10 8 M, <5x10 M, <2x10 9 M, <109 M, about I1-0 M, 1040 M to 10-85 M, or 104 M to 10-M) as measured by plasmon resonance binding assay. The binding affinity can be represented by KD value, which is calculated as the ratio of dissociation rate to association rate (kr/ko, 1

) when the binding between the antigen and the antigen-binding molecule reaches equilibrium. The antigen-binding affinity (e.g. KD) can be appropriately determined using suitable methods known in the art, including, for example, plasmon resonance binding assay using instruments such as Biacore (see, for example, Murphy, M. et al, Current protocols in protein science, Chapter 19, unit 19.14, 2006).

[001181 In certain embodiments, the antibodies and the fragments thereof provided herein binds to human PD-Li with an EC5 o (i.e. 50% binding concentration) of0.nM-IOOnM (e.g. 0 inM-50nM, 0.InM-3nM, 0.nM-20nM, 0.nM-i10nM, or 0inmM-inM. Binding of the antibodies to human PD-LI can be measured by methods known in the art, for example, sandwich assay such as ELISA, Western Blot, FACS or other binding assay. In an illustrative example, the test antibody (i.e. first antibody) is allowed to bind to immobilized human PD-Li or cells expressing human PD-iL, after washing away the unbound antibody, a labeled secondary antibody is introduced which can bind to and thus allow detection of the bound first antibody. The detection can be conducted with a microplate reader when immobilized PD-1 is used, or by using FACS analysis when cells expressing human PD-LI are used. In certain embodiments, the antibodies and the fragments thereof provided herein binds to human PD-L1 with an EC.o (i.e. 50% effective concentration) of InM to 1OnM, or InM to 5nM as measured by FACS analysis.

[001191 In certain embodiments, the antibodies and the fragments thereof provided herein inhibit the binding of human PD-Li to its receptor at an IC5 0 of .2nM-IO0nM (e.g. 0.2nM 50nM, 0.2nM-30nM, 0.2nM-20nM, 0.2nM-l0nM or inM-lOnM), as measured in a competition assay.

1001201 In certain embodiments, the antibodies and the fragments thereof provided herein block binding of human PD-Li to its receptor and thereby providing biological activity including, for example, inducing cytokine production from the activated T cells (such as CD4+ T cells and CD8+ T cells), inducing proliferation of activatedT cells (such as CD4+ T cells and CD8+ T cells), and reversing T reg's suppressive function. Exemplary cytokines include IL-2 andINy. The term "IL-2" refers to interleukin 2, a type of cytokine signaling molecule in the immune system that regulates the activities of white blood cells (e.g. leukocytes). The terin "Interferon gamma (IFN)" is a cytokine that is produced by natural killer (NK), NK T cells, CD4* and CD8*T cells, which is a critical activator of macrophages and inducer of major histocompatibility complex (MI-IC) molecule expression. The cytokine production can be determined using methods known in the art, for example, by ELISA. Methods can also be used to detect proliferation of T cells, including [3H] thymidine incorporation assay.

1001211 The anti-PD-Li antibodies and the antigen-binding fragments thereof are specific for human PD-LI. In certain embodiments, the antibodies and antigen-binding fragments thereof do not bind to PD-L2 (e.g. human PD-L2). For example, the binding affinity with PD-L2 is less than 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that with human PD-L1.

1001221 In certain embodiments, the antibodies and antigen-binding fragments thereof bind to monkey PD-Li at an EC50 of no more than I0OnM, for example, no more than or about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 ,nM,1 nM ,0 8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM, or 0.0nM, as measured by ELISA. In certain embodiments, the antibodies and antigen-binding fragments thereof bind to monkey PD-Li at an EC50 of about 1 nM -1OnM.

[001231 In certain embodiments, the antibodies and antigen-binding fragments thereof do not bind to mouse PD-1. but bind to monkey PD-L1 with a binding affinity similar to that of human PD-Li. For example, binding of the exemplary antibodies 1.4.1, 1.14.4, 1.20.15, and 1.46.11 to mouse PD-i1 is not detectable in conventional binding assays such as ELISA, or FACS analysis, whereas the binding of these antibodies to monkey PD-L1 is at a similar affinity or EC50 value to that of human PD-Li as measured by ELISA or FACS.

[001241 In some embodiments, the anti-PD-LI antibodies and the antigen-binding fragments thereof has reduced or depleted effector function. In some embodiments, the anti-PD-LI antibodies arid the antigen-binding fragments thereof have a constant region of IgG4 isotype, which has reduced or depleted effector function. Effector functions such as ADCC and CDC can lead to cytotoxicity to cells expressing PD-1. Many cells including normal cells could express PD-L1. In order to avoid potential unwanted toxicity to those normal cells, certain embodiments of the antibodies and antigen-binding fragments provided herein can possess reduced or even depleted effector functions. Various assays are known to evaluate ADCC or CDC activities, for example, Fc receptor binding assay, Ciq binding assay, and cell lysis assay, and can be readily selected by people in the art. Without wishing to be bound to theory, but it is believed that antibodies with reduced or depleted effector functions such as ADCC or CDC would cause no or minimal cytotoxicity to PD-L-expressing cells, for example those normal cells, and therefore spare them from unwanted side effects, whereas in the meantime, tumor cells expressing PD-Li would be bound by the anti-PD-L1 antibodies and therefore cannot escape from the immune checkpoint and hence can be recognized and eliminated by the immune system.

[001251 In certain embodiments, the anti-PD-LI antibodies and antigen-binding fragments thereof provided herein have reduced side effects. For example, the antibodies and antigen binding fragments thereof provided herein can have fully human IgG sequence and therefore reduced immunogenicity than a humanized antibody. For another example, the antibodies and antigen-binding fragments thereof provided herein can be in IgG4 format to eliminate ADCC and CDC.

[001261 In certain embodiments, the anti-PD-LI antibodies and antigen-binding fragments thereof provided herein are advantageous in that they can be used in combination with immunogenic agents, such as tumor cells, purified tumor antigen, and cells transfected with genes encoding immune stimulating cytokines, tumor vaccines. In addition, the anti-PD-L1 antibodies and antigen-binding fragments thereof can be included in combination therapies, including standard chemo- and radio- therapies, target based small molecule therapies,. emerging other immune checkpoint modulator therapies. In certain embodiments, the antibodies and antigen-binding fragments thereof can be used as the base of antibody-drug conjugates, bispecific or multivalent antibodies.

[001271 The anti-PD-Li antibodies or antigen-binding fragments thereof provided herein can be a monoclonal antibody, polyclonal antibody, fully human antibody, humanized antibody, chimeric antibody, recombinant antibody, bispecific antibody, labeled antibody, bivalent antibody, or anti-idiotypic antibody. A recombinant antibody is an antibody prepared in vitro using recombinant methods rather than in animals. A bispecific or bivalent antibody is an artificial antibody having fragments of two different monoclonal antibodies and can bind two different antigens. An antibody or antigen-binding fragment thereof that is "bivalenf' comprises two antigen-binding sites. The two antigen binding sites may bind to the same antigen, or they may each bind to a different antigen, in which case the antibody or antigen-binding fragment is characterized as "bispecific."

[001281 In some embodiments, the anti-PD-LI antibodies or antigen-binding fragments thereof provided herein are fully human antibodies. In certain embodiments, the fully human antibodies are prepared using recombinant methods. For example, transgenic animal such as a mouse can be made to carry transgenes or transchromosomes of human immunoglobulin genes, and therefore capable of producing fully human antibodies after immunization with proper antigen such as human PD-Ll. Fully human antibodies can be isolated from such transgenic animal, or alternatively, can be made by hybridoma technology by fusing the spleen cells of the transgenic animal with an immortal cell line to generate hybridoma cells secreting the fully human antibodies. Exemplary transgenic animals include, without limitation, OmniRat, whose endogenous expression of rat immunoglobulin genes are inactivated and at the same time engineered to contain functional recombinant human immunoglobulin loci; OmniMouse, whose endogenous expression of mouse immunoglobulin genes are inactivated and at the same time engineered to contain recombinant human immunoglobulin loci having J-locus deletion and a C-kappa mutation; OmniFlic, which is a transgenic rat whose endogenous expression of rat immunoglobulin genes are inactivated and at the same time engineered to contain recombinant human immunoglobulin loci having a single common, rearranged VkJk light chain and functional heavy chain. Detailed information can be further found at: Osborn M. et al, Journal of Immunology, 2013, 190: 1481-90; Ma B et at, Journal ofImmunological Methods 400 - 401 (2013) 78-86; Geurts A. et al, Science, 2009, 325:433; U.S. Pat. 8,907,157; EP patent 2152880B1; EP patent 2336329131, all of which are incorporated herein by reference to its entirety. Other suitable transgenic animals can also be used, for example, HuMab mice (see, for details, Lonberg, N. et al. Nature 368(6474): 856 859 (1994)), Xeno-Mouse (Mendez et al. Nat Genet., 1997, 15:146-156), TransChromo Mouse (Ishida et al. Cloning Stem Cells, 2002, 4:91-102) and Velocimmune Mouse (Murphy et al. Proc Natl Acad Sci USA, 2014, 111:5153-5158), Kymouse (Lee et al. Nat Biotechnol, 2014, 32:356---363), and transgenic rabbit (Flisikowska et al. PLoS One, 2011, 6:e21045).

[001291 In some embodiments, the anti-PD-Li antibodies and the antigen-binding fragments thereof is a camelized single domain antibody, a diabody, a scFv, an scFv dimer, a13sFv, a dsFv, a (dsFv)2, a dsFv-dsFv', an Fv fragment, a Fab, a Fab', a F(ab)2, a ds diabody, a nanobody, a domain antibody, or a bivalent domain antibody.

[001301 In some embodiments, the anti-PD-Li antibodies and the antigen-binding fragments thereof further comprise an immunoglobulin constant region. In some embodiments, an immunoglobulin constant region comprises a heavy chain and/or a light chain constant region. The heavy chain constant region comprises CHI, CH1-CH2, or CH-CH3 regions. In some embodiments, the constant region may further comprise one or more modifications to confer desirable properties. For example, the constant region may be modified to reduce or deplete one or more effector functions, to improve FcRn receptor binding, or to introduce one or more cysteine residues.

[001311 In some embodiments, the anti-PD-LI antibodies and the antigen-binding fragments thereof further comprise a conjugate. It is contemplated that a variety of conjugates may be linked to the antibodies or antigen-binding fragments provided herein (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugates may be linked to the antibodies or antigen-binding fragments by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods. In certain embodiments, the antibodies and antigen-binding fragments disclosed herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugates. For example, such a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate. In certain embodiments, the antibodies may be linked to a conjugate indirectly, or through another conjugate. For example, the antibody or antigen binding fragments may be conjugated to biotin, then indirectly conjugated to a second conjugate that is conjugated to avidin. The conjugate can be a detectable label, a pharmacokinetic modifying moiety, a purification moiety, or a cytotoxic moiety. Examples of detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase. lysozyme, saccharide oxidases or f-D galactosidase), radioisotopes (e.g. 1241 us5,ruJ,3 S,H, "Inn, 2'In,' 4 C, 64 Cu, 6 Cu, 6 Y 8 8 r 9 0 yr 1 7 21 186C 188Re, mSm, 21 Bi, and "P, other lanthanides, luminescent labels), chromophoric moiety, digoxigenin, biotin/avidin, a DNA molecule or gold for detection. In certain embodiments, the conjugate can be a pharmacokinetic modifying moiety such as PEG which helps increase half-life of the antibody. Other suitable polymers include, such as, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like. In certain embodiments, the conjugate can be a purification moiety such as a magnetic bead. A "cytotoxic moiety" can be any agent that is detrimental to cells or that can damage or kill cells. Examples of cytotoxic moiety include, without limitation, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin done, mitoxantrone, mithramycin, actinomycin D, 1 dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

[001321 Polynucleotides and Recombinant Methods

1001331 The present disclosure provides isolated polynucleotides that encode the anti-PD Li antibodies and the antigen-binding fragments thereof In certain embodiments, the isolated polyncleotides comprise one or more nucleotide sequences as shown in Table 1, which encodes the CDR sequences provided in Table 1.

[001341 In some embodiments, the isolated polynucleotides encodes a heavy chain variable region and comprise a sequence selected from the group consisting of: SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, and ahomologous sequence thereof having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%) sequence identity. In some embodiments, the isolated polynucleotides encodes a light chain variable region and comprise a sequence selected from the group consisting of: SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, and a homologous sequence thereof having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity. In certain embodiments, the percentage identity is due to genetic code degeneracy, while the encoded protein sequence remains unchanged.

[00135] The isolated polynucleotide that encodes the anti-PD-Li antibodies and the antigen binding fragments thereof (e.g. including the sequences in Table 1) can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art. In another embodiment, the antibody may be produced by homologous recombination known in the art. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication., one or more marker genes, an enhancer element, a promoter (e.g. SV40, CMV, EF-l), and a transcription termination sequence.

[00136] In some embodiments, the vector system includes mammalian, bacterial, yeast systems, etc, and comprises plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO,pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBIpl5TV-L, pPro8,pTD,pRS420,pLexApACT2.2 etc, and other laboratorial and commercially available vectors. Suitable vectors may include, plasmid, or viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses).

[00137] Vectors comprising the polynucleotide sequence encoding the antibody or antigen binding fragment can be introduced to a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, eg.,Slmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

[00138] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-PD-Li antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (El 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[001391 Suitable host cells for the expression of glycosylated antibodies or antigen-fragment provided here are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruiffly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the Virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.

[001401 However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mramialian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, Proc. NatL Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243 251 (1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MIMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In some preferable embodiments, the host cell is 293F cell.

[001411 Host cells are transformed with the above-described expression or cloning vectors for anti-PD-LI antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[001421 The host cells used to produce the antibodies or antigen-binding fragments provided herein may be cultured in a variety of media. Commercially available media such as Ham's FIO (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEIVIM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655;or 5,122,469; WO 90/03430; WO 87/00195; orUSPat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYC[INL" drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[001431 When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such asP1MSFmay be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

[001441 The antibody prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human gamma., .gamma.2, or gamma.4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human .gamma.3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinvl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX.TM. resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPI-IAROSEm chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[001451 Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elation buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).

[001461 Kits

[001471 The present disclosure provides kits comprising the anti-PD-Li antibodies or the antigen-binding fragments thereof In some embodiments, the kits are useful for detecting the presence or level of PD-LI in a biological sample. The biological sample can comprise a cell or a tissue.

[001481 In some embodiments, the kit comprises nanti-PD-Li antibody or the antigen binding fragment thereof which is conjugated with a detectable label. In certain other embodiments, the kit comprises an unlabeled anti-PD-LIantibody or antigen-binding fragment, and further comprises a secondary labeled antibody which is capable of binding to the unlabeled anti-PD-Liantibody. The kit may further comprise an instruction of use, and a package that separates each of the components in the kit.

[001491 In certain embodiments, the anti-PD-LI antibody or the antigen-binding fragment thereof are associated with a substrate or a device useful in a sandwich assay such as ELISA, or in an immunographic assay. Useful substrate or device can be, for example, microtiter plate and test strip.

1001501 Pharmaceutical Composition and Method ofTreatment

[001511 The present disclosure further provides pharmaceutical compositions comprising the anti-PD-L1 antibodies or the antigen-binding fragments thereof and one or more pharmaceutical acceptable carriers.

[001521 Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspendingldispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[001531 Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, inclusion of one or more antioxidants such as methionine in a composition comprising an antibody or antigen-binding fragment and conjugates as provided herein decreases oxidation of the antibody or antigen binding fragment. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf-life. Therefore, in certain embodiments compositions are provided that comprise one or more antibodies or antigen binding fragments as disclosed herein and one or more antioxidants such asmethionine. Further provided are methods for preventing oxidation of, extending the shelf-life of, and/or improving the efficacy of an antibody or antigen-binding fragment as provided herein by mixing the antibody or antigen-binding fragment with one or more antioxidants such as methionine.

[001541 To furtherillustrate, pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[001551 The pharmaceutical compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.

1001561 In embodiments, the pharmaceutical compositions are formulated into an injectable composition. The injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion. Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions. The solutions may be either aqueous or nonaqueous.

[001571 In certain embodiments, unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.

[001581 In certain embodiments, a sterile, lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides a desirable formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial can contain a single dosage or multiple dosages of the anti PD-Li antibody or antigen-binding fragment thereof or composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g., about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing. The Iyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.

[001591 Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution the sterile and/or non-pyretic water or other liquid suitable carrier is added to yophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.

[001601 Therapeutic methods are also provided, comprising: administering a therapeutically effective amount of the antibody or antigen-binding fragment as provided herein to a subject in need thereof, thereby treating or preventing a condition or a disorder associated with relatedtoPD-1. In another aspect, methods are provided to treat a condition in a subject that would benefit from upregulation of immune response, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment as provided herein to a subject in need thereof.

[00161] The therapeutically effective amount of an antibody or antigen-binding fragment as provided herein will depend on various factors known in the art, such as for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of tumor development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.

[00162] In certain embodiments, an antibody or antigen-binding fragment as provided herein may be administered at a therapeutically effective dosage of about 0.01 mg/kg to about 100 mg/kg(e.g., about 0.01 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg). In certain of these embodiments, the antibody or antigen-binding fragment is administered at a dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage is 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less. In certain embodiments, the administration dosage may change over the course of treatment. For example, in certain embodiments the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.

[001631 Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

[001641 The antibodies and antigen-binding fragments disclosed herein may be administered by any route known in the art, such as for example parenteral (e.g, subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[001651 Conditions and disorders associated with PD-Li can be immune related disease or disorder. in certain embodiments, the PD-L1 associated conditions and disorders include, tumors and cancers, for example, non-small cell lung cancer, small cell lung cancer, renal cell cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas, mycoses fungoids, merkel cell cancer, and other hematologic malignancies, such as classical Hodgkin lymphoma (CIL),primary mediastinal large B-cell lymphoma, T-cel1/histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD, and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma, and -- V8-associated primary effusion lymphoma, Hodgkin's lymphoma, neoplasm of the central nervous system (CNS), such as primary CNS lymphoma, spinal axis tumor, brain stem glioma. In certain embodiments, the tumors and cancers are metastatic, especially metastatic tumors expressing PD-LI. In certain embodiments, the PD-LIassociated conditions and disorders include autoimmune diseases, such as systemic lupus erythematosus (SLE), psoriasis, systemic scleroderma, autoimmune diabetes and the like, In certain embodiments, the PD-Li associated conditions and disorders include infectious disease such as chronic viral infection for example, viral infection of hepatitis B, hepatitis C, herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type2, human papilloma virus, adenovirus, Kaposi West sarcoma associated herpes virus epidemics, thin ring virus (Torquetenovirus), JC virus or BK virus.

[001661 Methods of Use

[001671 The present disclosure further provides methods of using the anti-PD-Li antibodies or the antigen-binding fragments thereof

1001681 In some embodiments, the present disclosure provides methods of treating a condition or a disorder associated with related to PD-Li in an individual, comprising administering a therapeutically effective amount of the anti-PD-Li antibody or antigen binding fragment thereof. In certain embodiments, the individual has been identified as having a disorder or condition likely to respond to a PD-Li antagonist.

1001691 The presence or level of PD-L1 on an interested biological sample can be indicative of whether the individual from whom the biological sample is derived could likely respond to a PD-Li antagonist. Various methods can be used to determine the presence or level of PD Li in a test biological sample from the individual. For example, the test biological sample can be exposed to anti-PD-LI antibody or antigen-binding fragment thereof, which binds to and detects the expressed PD-Li protein. Alternatively, PD-Li can also be detected at nucleic acid expression level, using methods such as qPCR, reverse transcriptase PCR, microarray, SAGE, FISH, and the like. In some embodiments, the test sample is derived from a cancer cell or tissue, or tumor infiltrating immune cells. In certain embodiments, presence or upregulated level of the PD-L in the test biological sample indicates likelihood of responsiveness. The term "upregulated" as used herein, refers to an overall increase of no less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80% or greater, in the protein level of PD-LI in the test sample as detected using the antibodies or antigen-binding fragments provided herein, as compared to the PD-LI protein level in a reference sample as detected using the same antibody. The reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non diseased sample obtained from the same individual from whom the test sample is obtained. For example, the reference sample can be a non-diseased sample adjacent to or in the neighborhood of the test sample (e.g. tumor).

[001701 Theantibodies orantigen-bindingfragments disclosed herein maybe administered alone or in combination with one or more additional therapeutic means or agents. For example, the antibodies or antigen-binding fragments disclosed herein may be administered in combination with chemotherapy, radiation therapy, surgery for the treatment of cancer (e.g., tumorectomy), one or more anti-emetics or other treatments for complications arising from chemotherapy, or any other therapeutic agent for use in the treatment of cancer or any medical disorder mediated by PD-Li. In certain of these embodiments, an antibody or antigen-binding fragment as disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments the antibody or antigen binding fragment and the additional therapeutic agent(s) inay be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment administered "in combination" with another therapeutic agent does not have to be administered simultaneously with or in the same composition as the agent. An antibody or antigen-binding fragment administered prior to or after another agent is considered to be administered"in combination" with that agent as the phrase is used herein, even if the antibody or antigen-binding fragment and second agent are administered via different routes. Where possible, additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians' Desk Reference2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN 1563634457; 57th edition (November 2002)) or protocols well known in the art.

[001711 In certain embodiments, the therapeutic agents can induce or boost immune response against cancer. For example. a tumor vaccine can be used to induce immune response to certain tumor or cancer. Cytokine therapy can also be used to enhance tumor antigen presentation to the immune system. Examples of cytokine therapy include, without limitation, interferons such as interferon-ci, -P, and -,colony stimulating factors such as macrophage-CSF, granulocyte macrophage CSF, and granulocyte-CSF, interleukins such IL 1, IL-Ia, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12, tumor necrosis factors such as TNT-a and TNF-. Agents that inactivate immunosuppressive targets can also be used, for example, TGF-beta inhibitors, IL-10 inihibitors, and Fas ligand inhibitors. Another group of agents include those that activate immune responsiveness to tumor or cancer cells, for example, those enhance T cel activation (e.g. agonist of T cell costimulatory molecules such as CTLA-4, ICOS and OX-40), and those enhance dendritic cell function and antigen presentation.

1001721 The present disclosure further provides methods of monitoring therapeutic response or disease progression in a subject treated with PD-L antagonist, comprising determining presence or level of PD-L1 in a test biological sample from the individual with the anti-PD Li antibody or antigen-binding fragment thereof. In certain embodiments, the methods further comprise comparing the PD-LI level in the test biological sample with the PD-LI level in a comparable sample previously obtained from the same individual, wherein reduction, or slowed or halted increase in the PD-L1 level in the test biological sample indicates positive therapeutic response or controlled disease progression. The comparable sample can be the same type of sample as the test sample, but has been obtained from the same individual before treatment, or during an earlier stage of the treatment.

[001731 The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.

1001741 EXAMPLE 1: Antibody hybridoma generation

[001751 1.1 Immunization: female OMT rats (obtained from Open Monoclonal Technology, Inc., PaloAlto,US, 8-10 weeks of age), were primed with 10ug of human PD-1 ECD protein in TiterMax via footpad injection, and then boosted every 3 days with PD-Li ECD protein in Aluminium Phosphate Gel Adjuvant via footpad injection, until ready for fusion. Anti-PD-L Iantibody serum titers were examined by ELISA or FACS every other week.

[001761 1.2 Cell fusion: Three days prior to fusion, animals received a final boost with 10 g of human PD-L IECD protein in PBS through intraperitoneal injection. On the day of fusion, lymph nodes were harvested and prepared to get single cell suspension. Obtained lymphocytes were mixed with myeloma cells (P3) at proper ratio. Cell mixture was washed and re-suspended at 2.0 x 106 cells/mL in ECF solution. The fusion was performed by using the BTX2000 electricity instrument.

[001771 1.3 Primary and secondary screen of hybridoma supernatants: after culturin for 7 14 days at 37°C, a portion of the hybridoma supernatant was examined by using Mirrorball analysis. Briefly, the hybridoma supernatant was diluted 5 times in 1X PBS. PD-LI expressing CHO-K Icells were mixed with the secondary fluorochrome labeled antibody and DraQ5. In each well of the 384-well plate, 20 pL of the cell mixture and 20 L of the diluted hybridoma supernatant sample were added and incubated for at least 2 hours at room temperature in the dark, until ready for analysis on a Mirrorball@ high sensitivity microplate cytometer. The positive hits were confirmed by FACS using the PD-Li expressing CHO-KI cells. The cells were stained with the hybridoma supernatant samples, followed by 2nd antibody staining with FITC conjugated Goat Anti-Mouse IgG Fc. Corresponding parental cell lines were used as negative controls. The stained cells were analyzed by using a BD Biosciences FACSCanto II and FlowJo Version software.

[001781 1.4 Subclone: the hybridoma cell lines with confirmed positive binding to PD-LI expressing cells were used for subcloning. Briefly, for each hybridoma cell line, cells were counted and diluted to give 5 or 1 cells per 200 L in cloning medium. Plate 200 [L/well into the 96-well plates. Plates were incubated at 37°C, 5% CO2 until ready for following analysis.

[00179] 1.5 Isotype test: the[LISA plates were coated with 50 UL/well ofgoat anti-rat IgG1, IgG2a, IgG2b, IgG3, IgA and IgM antibodies atI g/mL, respectively. After blocking, 50 p L of hybridoma supernatant samples were added into each well, and incubated at room temperature for 2 hours. The goat anti-rat kappa light chain-HRP was used as the detecting antibody. The color reaction was developed using TMB substrate for 10 minutes, and stopped by 2M HJC. The plates were then read at 450nm on an ELISA microplate reader.

[001801 1.6 Cell based binding assay: To examine the binding activity of the fully human antibodies to target, CHJ-Ki cells that express human PD-L or mature dendritic cells (mDCs) were stained with the fully human antibodies, followed by 2" antibody staining with FITC conjugated goat anti-human IgG Fc. Corresponding parental cell lines were used as negative controls. The stained cells were analyzed by using a BD Biosciences FACSCanto 11 and FlowJo Version software.

[001811 The C1 cells transfected with full-length human PD-L1 were stained with antibodies against human PD-L Ifrom rat hybridoma, followed by2 "d antibody staining with FITC conjugated goat anti-rat fgG Fe and analyzed by FACS. As shown in Figure 1, antibodies 1.4.1, 1.14.4, 1.20.15 and 1.46.11 specifically bound to PD-LI expressed on CHO cells with EC50 values of about lnM.

[001821 EXAM PLE 2: Change Fc portion and purification

1001831 The antibodies in the 293F cell culture supernatant were then purified by using the protein A affinity chromatography.

[001841 EXAMPLE3: Fully human antibody characterization

[001851 3.1 Competition assay by FACS: to examine whether the fully human antibodies can block the binding of PD-Li to PD-1, the CHO-K1 cells expressing human PD-L1 were incubated with various concentrations of the fully human antibodies at 4C for 1 hour. The unbound antibodies were washed away, and then the mouse Fe-tagged human PD-I was added to the cells. The binding of human PD-i toPD-L expressing cell was detected by using FITC-conjugated goat anti-mouse IgG, followed by the analysis using a BD Biosciences FACSCanto II and FlowJo Version software.

[001861 CHO cells expressing human PD-Li were incubated with different concentrations of the fully human antibodies (1.4.1, 1.14.4, 1.20.15 and 1.46.11). Then the mouse Fc-tagged human PD-i was added to the cells. The binding of human PD-1 to PD-LI expressing cell was detected by using FITC-conjugated goat anti-mouse IgG, followed by the FACS analysis. As shown in Figure 2, all the tested fully human PD-L Iantibodies blocked the PD- Ibinding to PD-L1 expressed on transfected CHO cells, and 1.14.4, 1.20.15 and 1.46.11 showed an IC 50 value of about I0nM.

[001871 3.2 Affinity test by surface plasmon resonance (SPR): Antibodies were characterized for affinity and binding kinetics to PD-Li by SPR assay using ProteOn XPR36 (Bio-Rad). Protein A protein (Sigma) was immobilized to a GLM sensor chip (Bio-Rad) through amine coupling. Purified antibodies were flowed over the sensor chip and captured by the Protein A. The chip was rotated 90° and washed with running buffer (IXPBS/ 0.01% Tween20, Bio-Rad) until the baseline is stable. Five concentrations of human PD-L1 and running buffer were flowed against the antibody flow cell at a flow rate 100 [L/min for an association phase of 240s, followed by 600s dissociation. The chip was regenerated with pH 1.7 H3PO4 after each run. The association and dissociation curve was fit to a 1:1 Langmiur binding model using ProteOn software.

[001881 As shown in Figure 5, the affinities of fully human PD-L1 antibodies for recombinant human PD-Liwere from 4.78E-10 to2.26E-10 mo/, as measured by surface plasmon resonance.

[001891 3.3 Affinity test by FACS: antibody binding affinity to cell surface PD-Li was performed by FACS analysis using CHO-KI cells expressing human PD-Li. Tested antibodies were I in 2 serially diluted in wash buffer (IXPBS/ 1%BSA) and incubated with cells at 4"°C for I h. The secondary antibody goat anti-human IgG Fc FITC (Jackson Immunoresearch Lab) was added and incubated at 4 °C in the dark for I Ii. The cells were then washed once and resuspended in IXPBS/I%BSA, and analyzed by flow cytometery (3D). Fluorescence intensity will be converted to bound molecules/cell based on the quantitative beads QuantumTM MESF Kits (Bangs Laboratories, Inc.). KD was calculated using Graphpad PrismS.

[001901 3.4 In vitro functional assay: to evaluate the ability of the fully human antibodies in modulatingT cell responsiveness, including the cytokine production and cell proliferation, following three assays were performed.

[001911 3.4.1 Allogeneic MLR: monocytes were isolated from healthy donors using Human Monocyte Enrichment kit according to the manufacturer's instruction. Cells were cultured for 7 days to differentiate into dendritic cells (DCs). 18 to 24 hours before usage, 1 g/ml LPS was added to the cell culture to induce the maturation of the DCs.

[001921 CD4* T cells were isolated usingHuman CD4TC ell Enrichment kit according to the manufacturer's protocol, and then were stimulated with the mature or immature allogenenic DCs in the presence or absence of fully human antibodies or control Ab. The levels of IL-2 and IFNy in the culturesupernatant were measured by ELISA on Day 3 and Day 5, respectively. The proliferation of CD4* T cells were assessed by [3H] thymidine incorporation.

1001931 As shown in Figure 9, all the tested fully human PD-LI antibodies (1.4.1, 1.14.4, 1.20.15 and 1.46.11) increased IL-2 secretion in a dose manner. As shown in Figure 8, all the tested fully human PD-Li antibodies (1.4.1, 1.14.4, 1.20.15 and 1.46.11) increased IFN secretion in a dose manner. As shown in Figure 10, all the tested fully human PD-LI antibodies (1.4.1, 1.14.4, 1.20 15 and 1.46.11) enhanced concentration dependent T cell proliferation.

[001941 3.4.2 Autologous Ag-specific immune response: PBMC and monocytes were isolated from the same donor. PBMC were cultured in the presence of CMV pp65 peptide and low dose of L2 (20U/ml). At the meantime, DCs were generated by culturing monocytes as previously mentioned. After 5 days, the DCswere pulsed with pp65 peptide and then added to the CD4* T cells in the presence or absence of the fully human antibodies or control Ab The levels of IL-2 and IFNy in the culture supernatant were measured by ELISA on Day 3 and Day 5, respectively. The proliferation of CMVpp65-specific CD4* T cells were assessed by [3-] thymidine incorporation.

[001951 As shown in Figure 6, the IFNy production in specific T cell response was enhanced by the fully human PD-L1 antibodies (1.4.1, 1.14.4, 1.20 15 and 1.46 11). Figure 7 shows that fully human PD-L1 antibodies enhanced concentration dependent CMV*- CD4-T cell proliferation stimulated with CMV pp65 peptide-loaded autologous DCs.

[001961 3.4.3 Treg suppression assay: regulatoryT cells (Tregs) are a key immune modulator and playkey roles in maintaining self-tolerance. CD4*CD25* Tregs are associated with tumors because increased numbers ofTregs were found in patients with multiple cancers and are associated with a poorer prognosis. To directly assess the effect of anti-human PD-LI fully human antibodies on Tregs' inhibitory function, we compared the Treg's function in the presence or absence of fully human antibodies or control Ab. Briefly, CD4*CD25* Tregs and CD4*CD25 T cells were separated by MACS. CD4-CD25Tregs and CD4CD25-T cells (Treg:Teff 1:Iratio) were co-cultured with allogeneic mDCs in the presence or absence of the fully human antibodies or control Ab at different concentrations. Either no antibody or isotype antibody was used as negative control. The cytokine production and T cell proliferation were measured as previously mentioned.

[001971 As shown in Figure 11, PD-Li antibody 1.20.15 abrogated Treg's suppressive function and restored responding T cell proliferation and IFNy secretion.

[001981 3.5 Antibody-dependent cell-mediated cytoxicity (ADCC) and complement dependent cytoxicity (CDC) assay: as the human PD-Li is expressed in a variety of cell types, and on both healthy and tumor cells, to minimize the undesired toxicity on healthy PD-L1* cells, the selected anti-PD-Li fully human antibodies were confirmed to have no ADCC and CDC function.

[001991 3.5.1 ADCC: target cells (mDCs) and various concentrations of fully human antibodies were pre-incubated in 96-well plates for 30min, then IL-2 activated PBMCs (effector) were added at the effector/target ratio of 50:1. The plates were incubated for 6 hours at 37C in a 5% CO 2 incubator. Target cell lysis was determined by cytotoxicity detection kit (Roche). Optical density was measured by Molecular Devices SpectraMax Me Plate Reader. Control hAb (IgGI) and control hAb (IgG4) were used as positive and negative controls, respectively.

1002001 Using IL-2- activated PBMCs as a source of natural killer (NK) cells and mDC expressing high levels of cell surface PD-Li as target cells, fully human PD-Li antibodies (1.4.1, 1.14.4, 1.20.15 and 1.46.11) did not mediate ADCC (Figure 12).

[002011 3.5.2 CDC: target cells (mDC), diluted human serum complement (Quidel-AI12) and various concentrations of fully human antibodies were mixed in a 96-well plate. The platewas incubated for4 hat 37°C in a 5% CO 2 incubator. Target cell lysis was determined by CellTiter glo (Promega-G7573). Rituxan (Roche) and human B lymphoma cell line Raji (CD20 positive) were used as positive control. As shown in Figure 13, fully human PD-L1 antibodies did not mediated CDC.

[002021 3.6 Binning test by FACS: To examine whether the fully human antibodies were in the same epitope bin as the benchmark antibody, the CHO-KI cells expressing human PD-Li were incubated with different concentrations of the fully human antibodies at 4°C for 1 hour. The unbound antibodies were washed away, and then the biotin-tagged control Ab was added to the cells. The binding of the biotin-tagged control Ab to the PD-LIexpressing cells was detected by using PE-conjugated streptavidin, followed by the analysis using a BD Biosciences FACSCanto II and FlowJo Version software.

[002031 The results for the binning test showed that the epitope on human PD-Li bound by the fully human PD-L1 antibodies (i.e. 1.4.1, 1.14.4, 1.20.15 and 1.46.11) was different from the existing PD-Li antibodies (i.e. benchmark antibody).

[002041 3.7 Cross-species binding assay: the cross-reactivity of the Ab to cynomolgus and murine PD-Li was measured by ELISA. Human, cyno and mouse PD-L1 were coated on ELISA plates, respectively. After blocking, fully human antibodies were added into the plate and incubated at room temperature for at least 2 hours. The binding of the antibodies to the coated antigens was detected byusing goat anti-human IgG Fc-HRP. The color reaction was developed usingTM13 substrate and stopped by 2M HCI. The ELISA plates were analyzed at 450nm using a Molecular Device M5e microplate reader.

[002051 As shown in Figure 4, the result of ELISA experiment demonstrated that the tested fully human PD-1 bound to cynomoIgus monkey PD-1 in a dose dependent manner. However, none of the tested antibodies (1.4.1, 1.14.4, 1.20.15 and 1.46.11) bound to murine PD-LI.

[002061 3.8 Cross-family binding assay by FACS: to examine the cross-family binding activity of the fully human antibodies, cells lines that express PD-L2 were stained with the fully human antibodies, followed by 2d antibody staining with FITC conjugated goat anti human IgG Fe. PD-L Iexpressing cells were used as positive control. Corresponding parental cell lines were used as negative controls. The stained cells were analyzed by using a131) Biosciences FACSCanto 11 and FlowJo Version software.

[002071 CHO cells transfected with PD-Li or PD-L2 were stained with fully human PD-LI antibodies and analysis by FACS. As shown in Figure 3, the fully human PD-L1 antibodies bound specifically to PD-L, but not to PD-L2 of PD-1 ligand family.

[002081 Example 4: Epitope mapping of the fully human antibody

[002091 To determine the epitopes the present antibody 1.14.4 provided herein, alanine scanning experiments on hPD-1 and the effect evaluation to antibody binding were conducted using 1. 14.4.

[002101 Alanine scanning experiments on hPD-Li were conducted and their effect to antibody binding was evaluated. Alanine residues on hPD-Liwere mutated to glycine codons, and all other residues were mutated to alanine codons. For each residue of the hPD-L1I extracellular domain (ECD), point amino acid substitutions were made using two sequential PCR steps. A pcDNA3.3-hPD-LIECD.His plasmid that encodes ECD of human PD-LI and a C-terminal His-tag was used as template, and a set of mutagenic primer was used for first step PCR using the QuikChange lightning multi site-directed mutagenesis kit (Agilent technologies, Palo Alto, CA). Dpn I endonuclease was used to digest the parental template after mutant strand synthesis reaction. In the second-step PCR, linear DNA expression cassette which composed of a CMV promoter, an extracellular domain (ECD) of PD-LI, a His-tag and a herpes simplex virus thymidine kinase (TK) polyadenylation was amplified and transiently expressed in HEK293F cells (Life Technologies, Gaithersburg,MD).

1002111 Monoclonal antibody 1.14.4 was coated in plates for ELISA binding assay. After interacting with the supernatant that contains quantified PD-L1 mutant or human/mouse PD LiECD.His protein (Sino Biological, China), HRIP conjugated anti-His antibody (1:5000;

Rockland Immunochemicals, Pottstown, PA) was added as detection antibody. Absorbance was normalized according to the average of control mutants. After setting an additional cutoff to the binding fold change (<0.55), the final determined epitope residues were identified.

[002121 The binding activities of the antibodies 1.14.4 to both human and murine PD-L1 were conducted (Figure 14). Our lead 1 14.4 was found binding to human PD-Li (Figure 14A), but no bound to mouse PD-L1 (Figure 1413).

[002131 The effect of 131 PD-LI point mutations on antibody binding was shown in Table 2. Checking the positions of all these residues on the hPD- Icrystal structures (PDB code 3B1K and 4ZQK) revealed that some amino acids (e.g. Gly159,Tyr160, Prol61) were unlikely to directly contact any antibodies. The observed binding reductions most probably resulted from the instability or even collapse of hPD-L1 structure after alanine substitutions. After setting an additional cutoff to the binding fold change (<0.55), the final determined epitope residues were listed in Table 3. They are 6 positions to 1 14.4.

1002141 All data in Table 3 were therefore mapped on the crystal structure of hPD-L1 to make a better visualization and comparison. (Figure 15).

[002151 Table 2. The effect of PD-LI point mutations on antibody binding

1.14A

PD-L1 .DAI fold change a SD #Residue R i113 0A1 M0 K 62 f.6 .0

P 161 0.53 0.017 T 37 0l.569 0.002 E 129 0t599 0041 G 709 0.442 0.019 D 469 0t603 0.024 L 50 0647 0.004 A 2 0.5632 0.010 T 110 0.562 0.004 p 133 0.56 0.011 G 95 0.662 0.019 N 131 0.603 0.000 L 81 0.614 0.005 A 118 0.622 0.010 G 128 0,632 0.000 V 133 0.664 0.007 1 64 0.76 0.009 N 131 0.680 0.003 Y 1 0.692 0.008 E 218 0.702 0.000 V 127 0.705 0.0212 E 72 0.747 0.012 I 224 0.749 0.048 V 232 0.756 0.003 K 189 0.759 0.021 V 218 0.732 0.000 L 127 0.735 0.006 E 71 0.748 0.025 V 225 0.749 0.045 T 201 0.756 0.012 K 189 0.759 0.003 V 17 0.792 0.0010 L 109 0.770 0.0061 E 205 0.801 0.010 F 19 0.80 0.015 T 201 0.81 0.002 N 35 0.87 0.000

E 215 0.801 0.015

S 34 0.813 0.006 K 105 0.816 0.004 H- 78 0.821 0.006

E 217 0.823 0.136 G 119 0.825 0.010 E 39 0.831 0.002 E 188 0.833 0.000 P 43 0.834 0.010 V 44 0.837 0.001 L 231 0.838 0.003 Y 56 0.840 0.002 Y 28 0.842 0.015 L 175 0.842 0.009 N 236 0.848 0.019 1 199 0.848 0.011 K 185 0.853 0.003 K 25 0.857 0.080 D 90 0.858 0.003 D 103 0.863 0.007 T 203 0.870 0.015 R 212 0.870 0.006 T 22 0.875 0.004 1 206 0.876 0.002 V 29 0.882 0.001 T 102 0.887 0.001 T 154 0.888 0.004 T 179 0.893 0.030 G 120 0.895 0.007 F 191 0.897 0.021 T 196 0.898 0.049 Q 91 0,906 0.016 P 24 0,907 0.009 E 31 0.908 0.033 K 89 0.913 0.009 1 54 0,918 0.045 N 96 0.924 0.007 R 82 0.934 0.003 Y 32 0.935 0.009 S 117 0.935 0.010 S 169 0.939 0.018 E 45 0.942 0.002 N 183 0.944 0.021 T 181 0.947 0.038 V 23 0.948 0.019 L 27 0.949 0.001 E 237 0.956 0.001 L 214 0.958 0.013 R 125 0.959 0.009 S 79 0.962 0.007 D 26 0.963 0.010 T 20 0.974 0.017 S 80 0.974 0.015 Q 66 0.979 0.016 A 18 0.979 0.011

Q 83 0.982 0.003 V 174 0.983 0.009 D 73 0.988 0.014 K 162 0.990 0.008 T 180 1.000 0.025 R 86 1.001 0.003 A 98 1.009 0.002 Q 77 1.011 0.002 M 59 1.012 0.006 Q 100 1.012 0.002 K 75 1.015 0.021 M 115 1.016 0.040 V 143 1.016 0.031 V 147 1.017 0.005 L 74 1.021 0.018 N 135 1.026 0.130 A 51 1.035 0.037 M 36 1.036 0.005 L 142 1.047 0.072 Q 47 1.051 0.030 K 124 1.056 0.004 H 69 1.056 0.003 K 41 1.071 0.036 K 46 1.085 0.029 L 88 1.102 0.017 Y 123 1.108 0.005 1 38 1,132 0.026 S 93 1.145 0.024 A 121 1,147 0.018 L 94 1,189 0.021 D 122 [.215 0.026 SFold change in binding is relative to the binding of several silent alanine substitutions.

[002161 Table 3. Identification of potential epitopes

PD-Li to 1.14,4 residue location E 58 C strand E60 C strand D 61 C strand K62 CC loop N 63 CC' loop R 113 F strand Cutoff. fold change<0.55

1002171 As shown in Figure 15, the hot-spot residues in charge of the hPD-LI binding all gathered in the C strand, CC' loop and F strands (Figure 15). Checking the positions of the residues on the hPD-1/hPD-L1 complex crystal structures (PDB code 4ZQK. 4A) revealed that these residues mainly located on A, C, F and G strands. The epitopes of 1.14.4 was mainly contributed by the residues on the C strands, which have direct overlap with the hPD-

1 and hPD-L Iinteraction site, indicating the mechanisms in terms of hPD-Li binding and

hPD-1 blocking.

[002181 Example 5: In vivo inhibition of fully human antibody hPD-Llto the tumor growth

[002191 In order to assess the inhibition of hPD-Li antibody to the tumor growth, x 5 cels/0.1 mL of MC38-B7H1 tumor cells were inoculated subcutaneously to the anterior right rib of 42 male B-hPD- humanized mice. When the tumor size reached about 100mm 3

, the mice were grouped (5 groups, 7 per group) and administered with agents as follows: Group 1: vehicle, Group 2: control antibodyBMK6 (see detailed description in W02011066389A), Group 3: . 14.4, 3mg/kg, Group 4: 1.14.4, 10mg/kg and Group5: 1.14.4,30mg/kg. All groups were administered via intraperitoneal injection once every two days with six consecutive administration. The animals were continually observed for another two weeks after the end of administration. The tumor volume and body weight were measured twice a week, and the relationships between the change of mouse body weight and period of administration, and the change of tumor volume and period of administration were recorded. At the end of the experiment, the ratio of the tumor volumes in therapeutic groups to vehicle group (T/C) and tumor growth inhibition (TGI) were calculated and analyzed statistically. T-test was performed with Graphpad Prism 5 and tumor volume was analyzed statistically. P<0.05 was considered to have significant difference.

[002201 Tumor volume was measured twice a week using vernier caliper for the long diameter and short diameter, and the formula for calculating the volume is: Tumor volume:= 0.5 long diameter x short diameter2 . Tumor growth rate was calculated based on the measuring results: T/C(%)= tumor volume of therapeutic group/ tumor volume of negative control group x100%. Tumorgrowthinhibition TGI(%)= [-(Ti-T0)/(Vi-V0)]x100, wherein Ti is the average tumor volume of the therapeutic group at day i, TO is the average tumor volume of the therapeutic group at day 0, Vi is the average tumor volume of the control group at day i, and VO is the average tumor volume of the control group at day 0.

[002211 Table 4. Anti-tumor effects of fully human PD-Li antibody 1.14.4 to MC38-B7HI1 xenograft of murine colon cancer hPD- Ihumanized mice.

Tumor Tic Tumor Number Body weight (g)a Group of Before After volume (%)after 25 growth Grupof Bfoe ftr after 25 days of inhibition animals administration administration days of insulation inculation (%) (mm3) Group: Vehicle 7 27.6±1.3 28.8±1.5 2359 -- -- -

Group 2: BMK6 7 28.3±0.5 29.3±0.7 1241 52.6% 49.7% 0.02

Group 3: 1.14.4,3mg/kg 7 28.0±0.5 29.7±0.5 949 40.2% 62.8% 0.02

Group 4: 1.14.4,10mg/kg 7 27.8±0.8 30.3±1.1 1416 60.0% 42.0% 0.09

Group 5: 1.14.4,30mg/kg 7 26.7±0.9 27.5±1.0 1115 47.3% 55.4% 0.04

Notes: mean SE; b in comparison to control

[00222] During the experiment, the body weight of the animals in each group did not show significant decrease (Table 4 and Figure 16), indicating that the test agents have good tolerability. After the 19 days of administration (i.e. 25 days after tumor cells inoculation), the tumor volume in the vehicle group reached 2359mm 3 , and compared with the vehicle group, the tumor volumes in the groups of high, mediate and low doses of antibody 1.14.4 showed significant decrease (average tumor volume were 949 mm 3 , 1416 mm 3 and 1115 mm 3 , respectively). All the three doses of antibody showed significant anti-tumor effects indicating by the TGI 62.8%, 42.0% and 55.4%, respectively (Table 4 and Figure 17). The control antibody BMK6 also showed significant anti-tumor effect (average tumor volume is 1241 mm 3, and TGI is 49.7%). Therefore, results showed that antibody 1.14.4 showed significant anti-tumor effect, and the inhibitions for all the groups of high, mediate and low doses were above 40%.

[00223] While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments), it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.

[00224] Throughout this specification, unless otherwise indicated, "comprise," "comprises," and "comprising," (and variants thereof) or related terms such as "includes" (and variants thereof), are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers.

[00225] Throughout this specification, reference to any advantages, promises, objects or the like should not be regarded as cumulative, composite, and/or collective, and should be regarded as preferable or desirable rather than stated as a warranty.

05367406W002-seql.txt SEQUENCE LISTING <110> WuXi Biologics (Shanghai) Co. Ltd. Open Monoclonal Technology, Inc <120> NOVEL ANTI-PD-L1 ANTIBODIES

<130> 053674-8006WO02 <160> 56 <170> PatentIn version 3.5

<210> 1 <211> 7 <212> PRT <213> Homo sapiens <400> 1

Ile Arg Thr Tyr Tyr Trp Gly 1 5

<210> 2 <211> 21 <212> DNA <213> Homo sapiens

<400> 2 attagaactt actactgggg c 21

<210> 3 <211> 16 <212> PRT <213> Homo sapiens

<400> 3 Tyr Ile Tyr Tyr Ser Gly Ser Thr Arg Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15

<210> 4 <211> 48 <212> DNA <213> Homo sapiens <400> 4 tatatctatt atagtgggag cacccgctac aacccgtccc tcaagagt 48

<210> 5 <211> 7 <212> PRT <213> Homo sapiens

<400> 5 Leu Ser Tyr Phe Phe Asp Tyr 1 5

<210> 6 <211> 21 <212> DNA <213> Homo sapiens Page 1

05367406W002-seql.txt <400> 6 cttagctact tctttgacta c 21

<210> 7 <211> 11 <212> PRT <213> Homo sapiens <400> 7

Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala Cys 1 5 10

<210> 8 <211> 33 <212> DNA <213> Homo sapiens <400> 8 tctggagata aattggggga taaatatgct tgc 33

<210> 9 <211> 7 <212> PRT <213> Homo sapiens

<400> 9 Gln Asp Thr Lys Arg Pro Ser 1 5

<210> 10 <211> 21 <212> DNA <213> Homo sapiens

<400> 10 caagatacca agcggccctc a 21

<210> 11 <211> 9 <212> PRT <213> Homo sapiens

<400> 11 Gln Ala Trp Asp Ser Gly Thr Val Ile 1 5

<210> 12 <211> 27 <212> DNA <213> Homo sapiens

<400> 12 caggcgtggg acagcggcac tgtgata 27

<210> 13 <211> 5 <212> PRT Page 2

05367406W002-seql.txt <213> Homo sapiens <400> 13 Ser Tyr Ala Met Ser 1 5

<210> 14 <211> 15 <212> DNA <213> Homo sapiens

<400> 14 agctatgcca tgagt 15

<210> 15 <211> 17 <212> PRT <213> Homo sapiens <400> 15 Gly Ile Ser Gly Ser Gly Gly Phe Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15

Gly

<210> 16 <211> 51 <212> DNA <213> Homo sapiens <400> 16 ggtattagtg gtagtggtgg tttcacttac tacgcagact ccgtgaaggg c 51

<210> 17 <211> 12 <212> PRT <213> Homo sapiens

<400> 17 Pro Pro Arg Gly Tyr Asn Tyr Gly Pro Phe Asp Tyr 1 5 10

<210> 18 <211> 36 <212> DNA <213> Homo sapiens

<400> 18 cctcctcgtg gatacaacta tggccctttt gactac 36

<210> 19 <211> 11 <212> PRT <213> Homo sapiens <400> 19

Page 3

05367406W002-seql.txt Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His 1 5 10

<210> 20 <211> 33 <212> DNA <213> Homo sapiens <400> 20 gggggaaaca acattggaag taaaagtgta cac 33

<210> 21 <211> 7 <212> PRT <213> Homo sapiens <400> 21

Asp Asp Ser Asp Arg Pro Ser 1 5

<210> 22 <211> 21 <212> DNA <213> Homo sapiens

<400> 22 gatgatagcg accggccctc a 21

<210> 23 <211> 11 <212> PRT <213> Homo sapiens

<400> 23 Gln Val Trp Asp Ser Ser Ser Asp His Val Val 1 5 10

<210> 24 <211> 33 <212> DNA <213> Homo sapiens <400> 24 caggtgtggg atagtagtag tgatcacgtg gta 33

<210> 25 <211> 7 <212> PRT <213> Homo sapiens

<400> 25 Ser Ile Ser Asn Tyr Trp Gly 1 5

<210> 26 <211> 21 <212> DNA <213> Homo sapiens Page 4

05367406W002-seql.txt <400> 26 agtattagta actactgggg c 21

<210> 27 <211> 16 <212> PRT <213> Homo sapiens <400> 27

Ser Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Pro Leu Lys Ser 1 5 10 15

<210> 28 <211> 48 <212> DNA <213> Homo sapiens <400> 28 agtatctatt atagtgggag cacgaactac aatccgcccc tcaagagt 48

<210> 29 <211> 7 <212> PRT <213> Homo sapiens

<400> 29 Leu Thr Tyr Tyr Phe Asp Tyr 1 5

<210> 30 <211> 21 <212> DNA <213> Homo sapiens

<400> 30 ctgacctact actttgatta c 21

<210> 31 <211> 11 <212> PRT <213> Homo sapiens

<400> 31 Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala Cys 1 5 10

<210> 32 <211> 33 <212> DNA <213> Homo sapiens

<400> 32 tctggagata aattggggga taaatatgct tgc 33

<210> 33 <211> 7 <212> PRT Page 5

05367406W002-seql.txt <213> Homo sapiens <400> 33 Gln Asp Ser Lys Arg Pro Ser 1 5

<210> 34 <211> 21 <212> DNA <213> Homo sapiens

<400> 34 caagatagca agcggccctc a 21

<210> 35 <211> 9 <212> PRT <213> Homo sapiens <400> 35 Gln Thr Trp Asp Ser Ser Thr Val Val 1 5

<210> 36 <211> 27 <212> DNA <213> Homo sapiens

<400> 36 cagacgtggg acagcagcac tgtggta 27

<210> 37 <211> 5 <212> PRT <213> Homo sapiens

<400> 37

Ser Tyr Ala Met Ser 1 5

<210> 38 <211> 15 <212> DNA <213> Homo sapiens <400> 38 agctatgcca tgagt 15

<210> 39 <211> 17 <212> PRT <213> Homo sapiens <400> 39

Gly Phe Ser Gly Ser Gly Phe Ile Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15

Page 6

05367406W002-seql.txt Gly

<210> 40 <211> 51 <212> DNA <213> Homo sapiens <400> 40 ggttttagtg gtagtggttt tattacatac tacgcagact ccgtgaaggg c 51

<210> 41 <211> 12 <212> PRT <213> Homo sapiens <400> 41

Pro Pro Arg Gly Tyr Asn Tyr Gly Pro Phe Asp Tyr 1 5 10

<210> 42 <211> 36 <212> DNA <213> Artificial Sequence

<220> <223> Recombinant <400> 42 cctcctcgtg gatacaacta tggccctttt gactac 36

<210> 43 <211> 117 <212> PRT <213> Homo sapiens

<400> 43 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ile Arg 20 25 30

Thr Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Thr Gly Leu Glu 35 40 45

Trp Met Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Arg Tyr Asn Pro Ser 50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95

Cys Ala Arg Leu Ser Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu Page 7

05367406W002-seql.txt 100 105 110

Val Thr Val Ser Ser 115

<210> 44 <211> 351 <212> DNA <213> Homo sapiens

<400> 44 cagctgcaac tgcaggagtc gggcccagga ctggtgaagc cttcggagtc cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc attagaactt actactgggg ctggatccgc 120 cagcccccag ggacggggct ggagtggatg gggtatatct attatagtgg gagcacccgc 180

tacaacccgt ccctcaagag tcgagtcacc atatccgtag acacgtccaa gaaccagttc 240 tccctgaagc tgagctctgt gaccgccgca gacacggctg tgtattactg tgcgagactt 300 agctacttct ttgactactg gggccaggga accctggtca ccgtctcctc a 351

<210> 45 <211> 106 <212> PRT <213> Homo sapiens

<400> 45

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala 20 25 30

Cys Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Met Val Ile Tyr 35 40 45

Gln Asp Thr Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Leu Ala Met 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Gly Thr Val Ile 85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105

<210> 46 <211> 318 <212> DNA <213> Homo sapiens <400> 46 tcctatgaac tgactcagcc accctcagtg tccgtgtccc caggacagac agccagcatc 60 Page 8

05367406W002-seql.txt acctgctctg gagataaatt gggggataaa tatgcttgct ggtatcagca gaagccaggc 120

cagtcccctg tgatggtcat ctatcaagat accaagcggc cctcagggat ccctgagcga 180 ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac cctggctatg 240

gatgaggctg actattattg tcaggcgtgg gacagcggca ctgtgatatt cggcggaggg 300 accaagctga ccgtccta 318

<210> 47 <211> 121 <212> PRT <213> Homo sapiens <400> 47

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Gly Ile Ser Gly Ser Gly Gly Phe Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Lys Pro Pro Arg Gly Tyr Asn Tyr Gly Pro Phe Asp Tyr Trp Gly 100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser 115 120

<210> 48 <211> 363 <212> DNA <213> Homo sapiens <400> 48 gaggtgcaac tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagttgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcaggt attagtggta gtggtggttt cacttactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaacctcct 300

cgtggataca actatggccc ttttgactac tggggccagg gaaccctggt caccgtctcc 360 Page 9

05367406W002-seql.txt tca 363

<210> 49 <211> 108 <212> PRT <213> Homo sapiens <400> 49 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95

Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105

<210> 50 <211> 324 <212> DNA <213> Homo sapiens

<400> 50 tcctatgtgc tgactcagcc accctcggtg tcagtggccc caggacagac ggccaggatt 60 acctgtgggg gaaacaacat tggaagtaaa agtgtacact ggtaccagca gaagccaggc 120 caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240 gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcacgt ggtattcggc 300 ggagggacca agctgaccgt ccta 324

<210> 51 <211> 117 <212> PRT <213> Homo sapiens <400> 51 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15

Page 10

05367406W002-seql.txt Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ile 20 25 30

Ser Asn Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45

Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Pro 50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Thr Lys Asn Gln Phe 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95

Cys Ala Arg Leu Thr Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Met Leu 100 105 110

Val Thr Val Ser Ser 115

<210> 52 <211> 351 <212> DNA <213> Homo sapiens

<400> 52 cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc agtattagta actactgggg ctggatccgc 120

cagcccccag ggaaggggct ggagtggatt gggagtatct attatagtgg gagcacgaac 180

tacaatccgc ccctcaagag tcgagtcacc atatccgtag acacgaccaa gaaccagttc 240 tccctgaagc tgagctctgt gaccgccgca gacacggctg tgtattactg tgcgagactg 300

acctactact ttgattactg gggccaggga atgctggtca ccgtctcctc a 351

<210> 53 <211> 106 <212> PRT <213> Homo sapiens <400> 53 Ser Tyr Asp Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala 20 25 30

Cys Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Leu Leu Val Ile Gln 35 40 45

Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Page 11

05367406W002-seql.txt 50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 70 75 80

Asp Glu Ala Asp Tyr Phe Cys Gln Thr Trp Asp Ser Ser Thr Val Val 85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105

<210> 54 <211> 318 <212> DNA <213> Homo sapiens

<400> 54 tcctatgacc tgactcagcc accctcagtg tccgtctccc caggacagac agccagcatc 60 acctgctctg gagataaatt gggggataaa tatgcttgct ggtatcagca gaagccaggc 120

cagtcccctt tgctggtcat ccagcaagat agcaagcggc cctcagggat ccctgcgcga 180 ttctctggct ccaactctgg gaacacagcc actctgacca tcagcgggac ccaggctatg 240

gatgaggctg actatttctg tcagacgtgg gacagcagca ctgtggtatt cggcggaggg 300

accaagctga ccgtccta 318

<210> 55 <211> 121 <212> PRT <213> Homo sapiens

<400> 55

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Gly Phe Ser Gly Ser Gly Phe Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Met Pro Pro Arg Gly Tyr Asn Tyr Gly Pro Phe Asp Tyr Trp Gly 100 105 110

Page 12

05367406W002-seql.txt Gln Gly Thr Leu Val Thr Val Ser Ser 115 120

<210> 56 <211> 363 <212> DNA <213> Homo sapiens <400> 56 gaggtgcagt tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttagc agctatgcca tgagttgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaggt tttagtggta gtggttttat tacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa tacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gatgcctcct 300 cgtggataca actatggccc ttttgactac tggggccagg gaaccctggt caccgtctcc 360 tca 363

Page 13

Claims

1. An isolated anti-PD-Li antibody or an antigen binding fragment thereof, comprising a) a heavy chain variable region comprising CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 17; and a light chain variable region comprising CDR1 of SEQ ID NO: 19, CDR2 of SEQ ID NO: 21, and CDR3 of SEQ ID NO: 23; or b) a heavy chain variable region comprising CDR1 of SEQ ID NO: 37, CDR2 of SEQ ID NO: 39, and CDR3 of SEQ ID NO: 41 and a light chain variable region comprising CDR1 of SEQ ID NO: 19, CDR2 of SEQ ID NO: 21, and CDR3 of SEQ ID NO: 23.

2. The antibody or an antigen binding fragment thereof of claim 1, comprising a) a heavy chain variable region comprising SEQ ID NO: 47; and a light chain variable region comprising SEQ ID NO: 49; or b) a heavy chain variable region comprising SEQ ID NO: 55; and a light chain variable region comprising SEQ ID NO: 49.

3. The antibody or an antigen binding fragment thereof of any one of the preceding claims, capable of specifically binding to human PD-Liat an Kd value no more than 10-8 M as measured by plasmon resonance binding assay; optionally which binds to monkey PD-Li at an EC50 of no more than 10 nM, or no more than 1 nM, and/or does not bind to mouse PD-LI; optionally capable of inhibiting binding of human or monkey PD-Li to its receptor at an IC50 of no more than 100 nM; optionally which does not bind to PD-L2. optionally which does not mediate ADCC or CDC or both; optionally which is a fully human monoclonal antibody, preferably wherein the fully human monoclonal antibody is produced by a transgenic rat; and yet optionally has the epitope comprises at least one of the following amino acid residues of PD-L: E58, E60, D61, K62, N63 and RI13.

4. An antibody or an antigen binding fragment thereof of any one of the preceding claims, capable of blocking binding of human PD-Li to its receptor and thereby providing at least one of the following activities: a) inducing production of IL-2 in CD4*T cells; b) inducing production of IFNy in CD4*T cells; c) inducing proliferation of CD4*T cells; and d) reversing Treg's suppressive function. preferably which is a camelized single domain antibody, a diabody, a scFv, an scFv dimer, a BsFv, a dsFv, a (dsFv)2, a dsFv-dsFv', an Fv fragment, a Fab, a Fab', a F(ab') 2 , a ds diabody, a nanobody, a domain antibody, or a bivalent domain antibody; preferably further comprising an immunoglobulin constant region.

5. The antibody or antigen-binding fragment thereof of any one of the preceding claims, further comprising a conjugate.

6. An isolated polynucleotide encoding the antibody or an antigen binding fragment thereof of any one of claims 1-5.

7. A vector comprising the isolated polynucleotide of claim 6.

8. A host cell comprising the vector of claim 7.

9. A method of expressing the antibody or antigen-binding fragment thereof of any one of claims 1-5, comprising culturing the host cell of claim 8 under the condition at which the polynucleotide of claim 6 is expressed.

10. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-5.

11. A method of detecting presence or level of human or monkey PD-Li in a biological sample, comprising exposing the biological sample to the antibody or antigen-binding fragment thereof of any one of claims 1-5, and determining the presence or level of human or monkey PD-Li in the sample; or identifying an individual having a disorder or a condition likely to respond to a PD-Li antagonist, comprising: determining presence or level of PD-Li in a test biological sample from the individual with the antibody or antigen-binding fragment thereof of any of claims I 5, wherein presence or upregulated level of the PD-Li in the test biological sample indicates likelihood of responsiveness, preferably further comprising administering a therapeutically effective amount of antibody or antigen-binding fragment thereof of any one of claims 1-5 to the individual; or monitoring therapeutic response or disease progression in a subject treated with a PD Li antagonist, comprising determining presence or level of PD-Li in a test biological sample from the individual with the anti-PD-Li antibody or antigen-binding fragment thereof of any one of claims 1-5.

12. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-5 and one or more pharmaceutically acceptable carriers.

13. An antibody or antigen-binding fragment thereof of any one of claims 1-5 for use in treating a condition that would benefit from upregulation of immune response, preferably wherein the condition is a cancer or a chronic viral infection.

14. A method of treating a disorder or condition that would benefit from upregulation of immune response, the method comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-5 to a subject, preferably wherein the condition is a cancer or a chronic viral infection

15. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-5 in manufacture of a medicament for treating a condition in a subject that would benefit from upregulation of immune response, preferably wherein the condition is a cancer or a chronic viral infection.

16. An antibody or antigen-binding fragment thereof of any one of claims 1-5 for use in treating an individual having a disorder or a condition likely to respond to a PD-Li antagonist, wherein a presence or level of PD-Li from a test biological sample from the individual indicates likelihood of responsiveness, preferably wherein the condition is a cancer or a chronic viral infection.

17. A method of treating an individual having a disorder or a condition likely to respond to a PD-Li antagonist, the method comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-5 to the individual, wherein a presence or level of PD-Li from a test biological sample from the individual indicates likelihood of responsiveness, preferably wherein the condition is a cancer or a chronic viral infection.

18. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-5 in manufacture of a medicament for treating an individual having a disorder or a condition likely to respond to a PD-Li antagonist, wherein a presence or level of PD-Li from a test biological sample from the individual indicates likelihood of responsiveness, preferably wherein the condition is a cancer or a chronic viral infection.